Patent application title: ENGINEERED MICROCOMPARTMENT PROTEIN AND RELATED METHODS AND SYSTEMS OF ENGINEERING BACTERIAL SYSTEMS FOR NON-NATIVE PROTEIN EXPRESSION AND PURIFICATION
Inventors:
Mimi Cho Yung (Milpitas, CA, US)
Timothy S. Carpenter (Livermore, CA, US)
Tek Hyung Lee (Pleasanton, CA, US)
David Savage (Berkeley, CA, US)
IPC8 Class: AC07K14195FI
USPC Class:
1 1
Class name:
Publication date: 2019-09-12
Patent application number: 20190276501
Abstract:
Engineered microcompartment proteins, and related engineered
microcompartment, vectors, cells compositions, methods and systems
configured to provide within a cell one or more proteins non-native to
the cell are described, wherein the one or more proteins non-native to
the cell are contained in at least one engineered microcompartment within
the cell.Claims:
1. An engineered microcompartment protein, comprising an encapsulin
protein having sequence SEQ ID NO: 1 or a sequence with at least 22%
sequence identity or at least 40% sequence similarity to SEQ ID NO:1, and
comprising a P-Domain (Peripheral Domain), an E-Loop (Elongated Loop) and
an A-Domain (Axial Domain), wherein the P-Domain comprises a first
fragment of the P-domain having an N-terminus and a C-terminus and
comprising residues configured to form, in a folded encapsulin protein, a
secondary structure comprising in a direction N-terminus to C-terminus a
4 to 26 residues alpha helix .alpha.1, linked to a 0 to 22 residues first
non-structured region, linked to a 4 to 11 residues alpha helix .alpha.2,
linked to a 3 to 9 residues beta strand .beta.1, linked to a 3 to 13
residues second non-structured region, a second fragment of the P-Domain
having an N-terminus and a C-terminus and comprising residues configured
to form, in a folded encapsulin protein, a secondary structure comprising
a direction N-terminus to C-terminus a 9 to 15 residues beta strand
.beta.4, linked to a 6 to 15 residues alpha helix .alpha.3, linked to a 0
to 10 residues first non-structured region, a 18 to 29 residues alpha
helix .alpha.4, and a 9 to 21 residues second non-structured region, and
a third fragment of the P-domain having an N-terminus and a C-terminus
and comprising residues configured to form, in a folded microcompartment
protein, a secondary structure comprising in a direction N-terminus to
C-terminus a 4 to 10 residues beta strand .beta.9, linked to a 3 to 16
residues first non-structured region, linked to a 7 to 13 residues beta
strand .beta.10, linked to a 1 to 15 residues second non-structured
region, linked to a 10 to 19 residues beta strand .beta.11; the E-Loop
has an N-terminus and a C-terminus and comprises residues configured to
form in a folded encapsulin protein, a secondary structure comprising in
a direction N-terminus to C-terminus a 8 to 16 residues beta strand
.beta.2, linked to a 2 to 24 residues first non-structured region, linked
to a 7 to 15 residues beta strand .beta.3, linked to a 0 to 6 residues
second non-structured region; and the A-Domain of the encapsulin protein
has an N-terminus and a C-terminus and comprises residues configured to
form in a folded encapsulin protein, a secondary structure comprising in
a direction N-terminus to C-terminus a 0 to 8 residues beta strand
.beta.5, linked to a 1 to 15 residues first non-structured region, linked
to a 16 to 23 residues alpha helix .alpha.5, linked to a 3 to 11 residues
second non-structured region, linked to a 3 to 11 residues beta strand
.beta.36, linked to a 9 to 16 residues alpha helix .alpha.6, linked to a
1 to 24 third non-structured region, linked to a 0 to 16 residues alpha
helix .alpha.7, linked to a 0 to 8 residues fourth non-structured region,
linked to a 1 to 10 residues beta strand .beta.7, linked to a 1 to 12
residues fifth non-structured region, linked to a 3 to 10 residues beta
strand .beta.8, linked to a 2 to 12 residues sixth non-structured region;
and wherein the P-domain, A-domain and E-loop are arranged together in a
configuration comprising, in a direction N-terminus to C-terminus, the
first fragment of the P-domain linked to the E-loop linked to the second
fragment of the P-domain linked to the A-domain linked to the third
fragment of the P-domain; the engineered microcompartment protein further
comprising, a target protein having an N-terminus, a C-terminus inserted
at the N-terminus of the first segment of the P-domain of the encapsulin
protein alone or in combination with a tag and/or a linker; at least one
first protease cleavage site inserted between the C-terminus of the
target protein and the N-terminus of the first segment of the P-Domain of
the encapsulin protein, alone or in combination with a tag and/or a
linker; and at least one second protease cleavage site inserted at an
insertion site at the C terminus of the E-loop of the encapsulin protein,
within 1 to 17 amino acids adjacent to the C-terminus of the E-loop of
the encapsulin protein and/or within 2-14 amino acids adjacent to the
N-terminus of the A-domain of the encapsulin protein, alone or in
combination with a tag and/or a linker to enable digestion of the
encapsulin and release of the target protein.
2. The engineered microcompartment protein of claim 1, wherein the target protein comprises at least one non-native antimicrobial peptide.
3. The engineered microcompartment protein of claim 2, wherein the at least one non-native antimicrobial peptide is selected from Apidaecin Ia, HBCM-2, cecropins, magainins, melittin, protegrins, and nisins.
4. The engineered microcompartment protein of claim 1, wherein the target protein has a sequence up to 80 amino acids in length.
5. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted within the first non-structured region of the E-loop to provide a cage-forming engineered microcompartment protein.
6. The engineered microcompartment of claim 5, wherein the at least one second protease cleavage site is inserted at positions X57 and/or X66 of SEQ ID NO: 1.
7. The engineered microcompartment of claim 6, wherein the E-loop has sequence YAAHPLGEVEVLSDENEVVKWGLRK SLP (SEQ ID NO: 59), YTVVPEGRLKKIEDNPGNVCTGMYQVKP (SEQ ID NO: 60), YAAVNTGELRPIDDTPEDVDMKLRQVQP (SEQ ID NO: 61), YAAVNT GRRT ALE DKAEGA S IF QRQVLP (SEQ ID NO: 62), or FSALGTGHVSRVAADTPGVEALQRHVVR (SEQ ID NO: 63).
8. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted in the first non-structured region of the E-loop, the (35 beta strand of the A-domain and/or the alpha helix cc5 of the A-Domain to provide a cage forming engineered microcompartment protein.
9. The engineered microcompartment of claim 8, wherein the at least one second protease cleavage site is inserted at any one of positions X132 and X144 of SEQ ID NO: 1.
10. The engineered microcompartment of claim 8, wherein the A-Domain has sequence LLSF EERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGH YPLEKRVEECLRGGKIITTPRIEDALVVSER (SEQ ID NO: 64), LREGTSNPKLALPSSASDYPAAIAAALNQLRLAGVNGPYAVVLGAGVYTALSGG DDEGYPVFRHIESLIDGKIVWAPAIEGGFVLSTR (SEQ ID NO: 65) LLTEDGIVKFPISNWSEGENPFKDISIGLAKFIENGIVGRKALVVSPNLFVQLQRIQ PGTGTTEYDRINKLLDGNIFSTPVLKDDKAVLVCSE (SEQ ID NO: 66), ILNAEGAQKLQISDWGQGENPYTDIVKAINMIREKGIVGRFVLCLSQSLYFDLQRI QQGTGMTEAQRISSMIGNLYNVPVIKGKKAALICAE (SEQ ID NO: 67), or LLTVKGSSKIKKSDWSQGENSFADITAGVAQLAKTGYLGRYALVVSPDLFLDLQ RLQPNTGLLEIDRIKKLIGDNVYMTSVMGPGKAVLVCAE (SEQ ID NO: 68).
11. The engineered microcompartment of claim 5, wherein the target protein is a protease sensitive target protein.
12. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted within the beta-strand .beta.3 to provide a non-cage-forming engineered microcompartment protein.
13. The engineered microcompartment of claim 12, wherein the at least one second protease cleavage site is inserted at position X71 of SEQ ID NO: 1
14. The engineered microcompartment of claim 1, wherein the target protein is a protease sensitive target protein and the engineered microcompartment protein further comprises a proline between the N-terminus of the first at least one protease cleavage site and the C terminus of the target protein
15. The engineered microcompartment of claim 1, wherein the at least one second protease cleavage site is inserted at any one of positions X57, X60, X71 and X139.
16. The engineered microcompartment protein of claim 1 wherein the at least one encapsulin protein has a sequence of SEQ ID NO: 47.
17. The engineered microcompartment protein of claim 1, wherein the at least one encapsulin protein is from the PF04454 protein family.
18. The engineered microcompartment protein of claim 1, wherein the at least one first protease cleavage site and/or the at least one second protease cleavage site are selected from ENLYFQ\S(orG)) (TEV protease recognition), LEVLFQGP (HRV 3C protease recognition), LVPRGS (thrombin recognition), DDDDK (enterokinase recognition), and IEGR (Factor Xa recognition).
19. The engineered microcompartment protein of claim 1, wherein the target protein is fused to the encapsulin protein in an insertion region further comprising a linker and/or a tag.
20. The engineered microcompartment protein of claim 1, wherein the at least one first protease cleavage site is fused to the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein in an insertion region further comprising a linker and/or a tag.
21. The engineered microcompartment protein of claim 1, wherein the at least one second protease cleavage site is fused to the C-terminus of the E-loop and/or to the N-terminus of the A-domain of the encapsulin protein in an insertion region further comprising a linker and/or a tag.
22. The engineered microcompartment protein of claim 1, wherein the tag is selected from His-Tag, Strep-Tag, FLAG-Tag, Avi-Tag, E-Tag, HA-Tag, Myc-Tag, and TC-Tag.
23. An engineered microcompartment comprising at least one engineered microcompartment proteins of claim 1.
24. A method to produce in a bacterial cell a protein non-native to the bacterial cell, the method comprising introducing into the bacterial cell at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1 in which the target protein is the protein non-native to the bacterial cell; wherein the at least one first polynucleotide is operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the bacterial cell; and wherein the introducing is performed to obtain expression in the bacterial cell of the at least one engineered microcompartment protein to obtain the protein non-native to the bacterial within at least one engineered microcompartment formed by the at least one engineered microcompartment protein.
25. The method of claim 24, wherein the engineered microcompartment protein is the cage forming engineered microcompartment protein of claim 5.
26. The method of claim 24, wherein the engineered microcompartment protein is the non-cage forming engineered microcompartment protein of claim 12.
27. The method of claim 24, wherein the protein non-native to the bacterial cell is a toxic non-native protein capable of causing a cell damage.
28. The method of claim 24, wherein the protein non-native to the bacterial cell is a non-native protein capable of being degraded within the bacterial cell.
29. A system to produce, in bacterial cell, a protein non-native to the bacterial cell, the system comprising at least one first polynucleotide encoding at least one engineered microcompartment protein of claim 1, wherein the target protein is the protein non-native to the bacterial cell, the at least one engineered microcompartment protein operatively linked to one or more first regulatory elements configured to enable the expression of the at least one engineered microcompartment protein in one or more bacterial cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different engineered microcompartment proteins to form at least one microcompartment within the one or more bacterial cell, the system further comprising at least one of: the one or more bacterial cells capable of expressing the at least one first polynucleotide to provide an expressed engineered microcompartment protein herein described; at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the protease cleavage sites of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and at least one protease capable of targeting the protease cleavage site of the engineered microcompartment protein to release the protein non-native to the bacterial cell from the engineered microcompartment protein in the bacterial cell.
30. A vector comprising at least one polynucleotide encoding for an engineered microcompartment protein of claim 1, alone or in combination with regulatory elements in accordance with the disclosure.
31. A bacterial cell comprising at least one engineered microcompartment of claim 23.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. provisional application No. 62/598,984, entitled "Engineered Microcompartment Protein and Related Methods and Systems of Engineering Bacterial Systems for Non-native Protein Expression and Purification" filed on Dec. 14, 2017, with docket number IL-13262, which is incorporated by reference in its entirety.
FIELD
[0003] The present disclosure relates to protein production in cell systems and in particular to engineering microcompartment proteins and related bacterial systems for non-native protein expression and purification and related cells, compositions, methods and systems.
BACKGROUND
[0004] Production of non-native proteins has been the subject of research in several fields, including commercial and academic fields in connection with applications involving production in a cell of a protein non-native to that cell.
[0005] Despite the presence of various approaches, expression, production and/or purification in a cell of proteins non-native to said cell for various uses is still challenging.
SUMMARY
[0006] Provided herein, are engineered microcompartment proteins and related engineered microcompartments, vectors, cells, compositions, methods and systems that can be used in several embodiments for non-native protein expression, production and/or purification.
[0007] According to a first aspect, an engineered microcompartment protein is described. The engineered microcompartment protein comprises an encapsulin protein having sequence
[0008] X.sub.1--X.sub.2--X.sub.3--X.sub.4--X.sub.5--X.sub.6--X.sub.7--X.sub.8--X- .sub.9--X.sub.10--X.sub.11--X.sub.12--X.sub.13--X.sub.14--X.sub.15--X.sub.- 16--X.sub.17--X.sub.18--X.sub.18--X.sub.20--X.sub.21--X.sub.22--X.sub.23--- X.sub.24--X.sub.25--X.sub.26--X.sub.27--X.sub.28--X.sub.29--X.sub.30 X.sub.31--X.sub.32--X.sub.33 X.sub.34--X.sub.35--X.sub.36--X.sub.37--X.sub.38--X.sub.39--X.sub.40--X.s- ub.41--X.sub.42--X.sub.43--X.sub.44--X.sub.45--X.sub.46--X.sub.47--X.sub.4- 8--X.sub.49--X.sub.50--X.sub.51--X.sub.52--X.sub.53--X.sub.54--X.sub.55--X- .sub.56--X.sub.57--X.sub.58--X.sub.59--X.sub.60--X.sub.61--X.sub.62--X.sub- .63--X.sub.64--X.sub.65--X.sub.66--X.sub.67--X.sub.68--X.sub.69--X.sub.70-- -X.sub.71--X.sub.72--X.sub.73--X.sub.74--X.sub.75--X.sub.76--X.sub.77--X.s- ub.78--X.sub.79--X.sub.80--X.sub.81--X.sub.82--X.sub.83--X.sub.84X.sub.85-- -X.sub.86--X.sub.88--X.sub.89--X.sub.90--X.sub.91--X.sub.92--X.sub.93--X.s- ub.94--X.sub.95--X.sub.96--X.sub.97--X.sub.98--X.sub.99--X.sub.100--X.sub.- 101--X.sub.102--X.sub.103--X.sub.104--X.sub.105--X.sub.106--X.sub.107--X.s- ub.108--X.sub.109--X.sub.110--X.sub.111--X.sub.112--X.sub.113--X.sub.114--- X.sub.115--X.sub.116--X.sub.117--X.sub.118--X.sub.119--X.sub.120--X.sub.12- 1--X.sub.122X.sub.123--X.sub.124--X.sub.125--X.sub.126--X.sub.127--X.sub.1- 28--X.sub.129--X.sub.130--X.sub.131--X.sub.132--X.sub.133--X.sub.134--X.su- b.135--X.sub.136--X.sub.137--X.sub.138--X.sub.139--X.sub.140--X.sub.141--X- .sub.142--X.sub.143--X.sub.144--X.sub.145--X.sub.146--X.sub.147--X.sub.148- --X.sub.149--X.sub.150--X.sub.151--X.sub.152--X.sub.153--X.sub.154--X.sub.- 155--X.sub.156--X.sub.157--X.sub.158--X.sub.159--X.sub.160--X.sub.161--X.s- ub.162--X.sub.163--X.sub.164--X.sub.165--X.sub.166--X.sub.167--X.sub.168--- X.sub.169--X.sub.170--X.sub.171--X.sub.172--X.sub.173--X.sub.174--X.sub.17- 5--X.sub.176--X.sub.177--X.sub.178--X.sub.179--X.sub.180--X.sub.181--X.sub- .182--X.sub.183--X.sub.184--X.sub.185--X.sub.186--X.sub.187--X.sub.188--X.- sub.189--X.sub.190--X.sub.191--X.sub.192--X.sub.193--X.sub.194--X.sub.195-- -X.sub.196--X.sub.197--X.sub.198--X.sub.199--X.sub.200--X.sub.201--X.sub.2- 02--X.sub.203--X.sub.204--X.sub.205--X.sub.206--X.sub.207--X.sub.208--X.su- b.209--X.sub.210--X.sub.211--X.sub.212--X.sub.213--X.sub.214--X.sub.215--X- .sub.216--X.sub.217--X.sub.218--X.sub.219--X.sub.220--X.sub.221--X.sub.222- --X.sub.223--X.sub.224--X.sub.225--X.sub.226--X.sub.227--X.sub.228--X.sub.- 229--X.sub.230--X.sub.231--X.sub.232--X.sub.233--X.sub.234--X.sub.235--X.s- ub.236--X.sub.237--X.sub.238--X.sub.239--X.sub.240--X.sub.241--X.sub.242--- X.sub.243 --X.sub.244--X.sub.245--X.sub.246--X.sub.247--X.sub.248--X.sub.2- 49 X.sub.250--X.sub.251--X.sub.252--X.sub.253 (SEQ ID NO: 1) in which X.sub.1 is M, X.sub.2 is D, X.sub.3 is N, X.sub.4 is L, X.sub.5 is K, X.sub.6 is R, X.sub.7 is E, X.sub.8 is L, X.sub.9 is A, X.sub.10 is P, X.sub.11 is L, X.sub.12 is T, X.sub.13 is E, X.sub.14 is E, X.sub.15 is A, X.sub.16 is W, X.sub.17 is A, X.sub.18 is E, X.sub.19 is I, X.sub.20 is D, X.sub.21 is E, X.sub.22 is E, X.sub.23 is A, X.sub.24 is R, X.sub.25 is E, X.sub.26 is T, X.sub.27 is A, X.sub.28 is K, X.sub.29 is R, X.sub.30 is H, X.sub.31 is L, X.sub.32 is A, X.sub.33 is G, X.sub.34 is R, X.sub.35 is R, X.sub.36 1S V, X.sub.37 1S V, X.sub.38 is D, X.sub.39 1S V, X.sub.40 is E, X.sub.41 is G, X.sub.42 is P, X.sub.43 is L, X.sub.44 is G, X.sub.45 1S W, X.sub.46 is G, X.sub.47 is Y, X.sub.48 is S, X.sub.49 is A, X.sub.50 1S V, X.sub.51 is P, X.sub.52 is L, X.sub.53 is G, X.sub.54 is R, X.sub.55 is L, X.sub.56 is E, X.sub.57 is E, X.sub.58 is I, X.sub.59 is E, X.sub.60 is G, X.sub.61 is P, X.sub.62 is A, X.sub.63 is E, X.sub.64 is G, X.sub.65 is V, X.sub.66 is Q, X.sub.67 is A, X.sub.68 is G, X.sub.69 is V, X.sub.70 is R, X.sub.71 is Q, X.sub.72 is V, X.sub.73 is L, X.sub.74 is P, X.sub.75 is L, X.sub.76 is P, X.sub.77 is E, X.sub.78 is L, X.sub.79 is R, X.sub.80 is V, X.sub.81 is P, X.sub.82 is F, X.sub.83 is T, X.sub.84 is L, X.sub.85 is 5, X.sub.86 is R, X.sub.87 is R, X.sub.88 is D, X.sub.89 is L, X.sub.90 is D, X.sub.91 is A, X.sub.92 is V, X.sub.93 is E, X.sub.94 is R, X.sub.95 is G, X.sub.96 is A, X.sub.97 is K, X.sub.98 is D, X.sub.99 is L, X.sub.100 is D, X.sub.101 is L, X.sub.102 is S, X.sub.103 is P, X.sub.104 is V, X.sub.105 is VA, X.sub.106 is E, X.sub.107 is A, X.sub.108 is A, X.sub.109 is R, X.sub.110 is L, X.sub.111 is L, X.sub.112 is A, X.sub.113 is R, X.sub.114 is A, X.sub.115 is E, X.sub.116 is D, X.sub.117 is R, X.sub.118 is L, X.sub.119 is I, X.sub.120 is F, X.sub.121 is N, X.sub.122 is G, X.sub.123 is Y, X.sub.124 is A, X.sub.125 is E, X.sub.126 is A, X.sub.127 is G, X.sub.128 is I, X.sub.129 is E, X.sub.130 is G, X.sub.131 is L, X.sub.132 is L, X.sub.133 is N, X.sub.134 is A, X.sub.135 is S, X.sub.136 is G, X.sub.137 is N, X.sub.138 is L, X.sub.139 is K, X.sub.140 is L, X.sub.141 is P, X.sub.142 is L, X.sub.143 is S, X.sub.144 is A, X.sub.145 is D, X.sub.146 is P, X.sub.147 is G, 3 X.sub.148 is D, X.sub.149 is I, X.sub.150 is P, X.sub.151 is D, X.sub.152 is A, X.sub.153 is I, X.sub.154 is A, X.sub.155 is E, X.sub.156 is A, X.sub.157 is L, X.sub.158 is T, X.sub.159 is K, X.sub.160 is L, X.sub.161 is R, X.sub.162 is E, X.sub.163 is A, X.sub.164 is G, X.sub.165 is V, X.sub.166 is E, X.sub.167 is G, X.sub.168 is P, X.sub.169 is Y, X.sub.170 is A, X.sub.171 is L, X.sub.172 is V, X.sub.173 is L, X.sub.174 is S, X.sub.175 is P, X.sub.176 is D, X.sub.177 is L, X.sub.178 is Y, X.sub.179 is T, X.sub.180 is A, X.sub.181 is L, X.sub.182 is F, X.sub.183 is R, X.sub.184 is V, X.sub.185 is Y, X.sub.186 is D, X.sub.187 is G, X.sub.188 is T, X.sub.189is G, X.sub.190 is Y, X.sub.191 is P, X.sub.192 is E, X.sub.193 is I, X.sub.194 is E, X.sub.195 is H, X.sub.196 is I, X.sub.197 is K, X.sub.198 is E, X.sub.199 is L, X.sub.200 is V, X.sub.201 is D, X.sub.202 is G, X.sub.203 is G, X.sub.204 is V, X.sub.205 is I, X.sub.206 is W, X.sub.207 is A, X.sub.208 is P, X.sub.209 is A, X.sub.210 is L, X.sub.211 is D, X.sub.212 is G, X.sub.213 is G, X.sub.214 is A, X.sub.215 is V, X.sub.216 is L, X.sub.217 is V, X.sub.218 is S, X.sub.219 is T, X.sub.220 is R, X.sub.221 is G, X.sub.222 is G, X.sub.223 is D, X.sub.224 is F, X.sub.225 is D, X.sub.226 is L, X.sub.227 is T, X.sub.228 is L, X.sub.229 is G, X.sub.230 is Q, X.sub.231 is D, X.sub.232 is L, X.sub.233 is S, X.sub.234 is I, X.sub.235 is G, X.sub.236 is Y, X.sub.237 is L, X.sub.238 is S, X.sub.239 is H, X.sub.240 is D, X.sub.241 is A, X.sub.242 is D, X.sub.243 is N, X.sub.244 is V, X.sub.245 is E, X.sub.246 is L, X.sub.247 is F, X.sub.248 is L, X.sub.249 is T, X.sub.250 is E, X.sub.251 is S, X.sub.252 is F, X.sub.253 is T (SEQ ID NO: 1)
[0009] or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO: 1.
[0010] In the engineered microcompartment protein, the encapsulin protein comprises a P-Domain (Peripheral Domain), an E-Loop (Elongated Loop) and an A-Domain (Axial Domain).
[0011] In the engineered microcompartment protein, the P-Domain of the encapsulin protein comprises
[0012] a first fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 26 residues alpha helix .alpha.l, linked to a 0 to 22 residues first non-structured region (such as a loop region), linked to a 4 to 11 residues alpha helix .alpha.2, linked to a 3 to 9 residues beta strand .beta.1, linked to a 3 to 13 residues second non-structured region.
[0013] a second fragment of the P-Domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded encapsulin protein, a secondary structure comprising a direction N-terminus to C-terminus a 9 to 15 residues beta strand (34, linked to a 6 to 15 residues alpha helix .alpha.3, linked to a 0 to 10 residues first non-structured region, a 18 to 29 residues alpha helix .alpha.4, and a 9 to 21 residues second non-structured region; and
[0014] a third fragment of the P-domain having an N-terminus and a C-terminus and comprising residues configured to form, in a folded microcompartment protein, a secondary structure comprising in a direction N-terminus to C-terminus a 4 to 10 residues beta strand .beta.9, linked to a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand .beta.10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand .beta.11.
[0015] In the engineered microcompartment protein, the E-Loop of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 8 to 16 residues beta strand .beta.2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand .beta.3, linked to a 0 to 6 residues second non-structured region.
[0016] In the engineered microcompartment protein, the A-Domain of the encapsulin protein has an N-terminus and a C-terminus and comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in a direction N-terminus to C-terminus a 0 to 8 residues beta strand .beta.5 linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix .alpha.5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand .beta.6, linked to a 9 to 16 residues alpha helix .alpha.6, linked to a 1 to 24 third non-structured region, linked to a 0 to 16 residues alpha helix .alpha.7, linked to a 0 to 8 residues fourth non-structured region, linked to a 1 to 10 residues beta strand .beta.7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand .beta.8, linked to a 2 to 12 residues sixth non-structured region.
[0017] In the engineered microcompartment protein, the P-domain, A-domain and E-loop are arranged together in a configuration comprising, in a direction N-terminus to C-terminus, the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain.
[0018] In the engineered microcompartment protein,
[0019] a target protein having an N-terminus, a C-terminus is inserted at the N-terminus of the first segment of the P-domain of the encapsulin protein alone or together with a linker and/or a tag
[0020] at least one first protease cleavage site is inserted between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein alone or together with a linker and/or a tag; and
[0021] at least one second protease cleavage site is inserted at the C-terminus of the E-loop of the encapsulin protein or within 1-17 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein, alone or together with a linker and/or a tag, to enable digestion of the encapsulin and release of the target protein.
[0022] In some embodiments wherein the engineered microcompartment proteins are designed to be non cage forming proteins, the at least one second protease cleavage site is inserted at the C-terminus of the E-loop of the encapsulin protein or within 1-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein (within .beta.3 of the E-loop).
[0023] In some embodiments wherein the engineered microcompartment proteins are designed for cage forming proteins, the at least one second protease cleavage site can be inserted within 9-17 amino acids from the C-terminus of the E-loop of the encapsulin protein (within the loop region between .beta.2 and .beta.3 of the E-loop), and/or inserted within 2-14 amino acids adjacent to the N-terminus of the A-domain (in the flexible region between the N-terminus of the A-domain and .alpha.5, including .beta.5).
[0024] In some embodiments, the target protein is a protein non-native to one or more bacterial cell (herein also indicated as non-native protein) and capable of causing cell damage (herein also indicated as non-native toxic protein). In some embodiments the at least one first protease and the at least one second protease cleavage site can be same or different.
[0025] According to a second aspect, an engineered microcompartment is described, the engineered microcompartment comprising a same or different at least one engineered microcompartment protein herein described. In particular, in some embodiments, the engineered microcompartment proteins of the engineered microcompartment have a same target protein, at least one first protease cleavage site and/or at least one second protease cleavage site.
[0026] According to a third aspect, a method is described to produce in a bacterial cell a protein non-native to the bacterial cell. The method comprises introducing into the bacterial cell at least one first polynucleotide encoding at least one engineered microcompartment protein herein described in which the target protein is the protein non-native to the bacterial cell. In the method, the at least one first polynucleotide is operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the bacterial cell. In the method the introducing is performed to obtain expression in the bacterial cell of the at least one engineered microcompartment protein to obtain the protein non-native to the bacterial within at least one engineered microcompartment formed by the at least one engineered microcompartment protein.
[0027] In some embodiments, the protein non-native to the bacterial cell is a toxic non-native protein capable of reacting with a native membrane substrate of the bacterial cell with a reaction resulting in a damage of the bacterial cell, and the engineered microcompartment protein is provided in the bacterial cell to shield the bacterial cell from toxicity during intracellular production of the toxic non-native protein in the bacterial cell.
[0028] According to a fourth aspect, a system is described to produce, in bacterial cell, a protein non-native to the bacterial cell. The system comprises
[0029] at least one first polynucleotide encoding at least one engineered microcompartment protein herein described wherein the target protein is the protein non-native to the bacterial cell, the at least one engineered microcompartment protein operatively linked to one or more first regulatory elements configured to enable the expression of the at least one engineered microcompartment protein in one or more bacterial cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different engineered microcompartment proteins to form at least one microcompartment within the one or more bacterial cell.
[0030] The system additionally comprises at least one of:
[0031] the one or more bacterial cells capable of expressing the at least one first polynucleotide to provide an expressed engineered microcompartment protein herein described;
[0032] at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and
[0033] at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the protein non-native to the bacterial cell from the engineered microcompartment protein in the bacterial cell.
[0034] In the system, the at least one first polynucleotide, the at least one second polynucleotide the at least one protease and the one or more bacterial cells are combined or simultaneously or sequentially used in the methods to produce in a bacterial cell a protein non-native to the bacterial cell herein described.
[0035] In some embodiments, the at least one non-native protein in the engineered microcompartment protein is a non-native toxic protein capable of causing cell damage, and the engineered microcompartment protein is provided in the cell to shield cell from toxicity during intracellular production of a toxic non-native protein.
[0036] According to a fifth aspect, a method is described to produce a non-native protein in a bacterial cell comprising at least one engineered microcompartment protein herein described in which the target protein is a protein non-native to the cell. The method comprises introducing into the bacterial cell at least one second polynucleotide encoding the at least one protease capable of cleaving the at least one first protease cleavage site and/or the at least one second protease cleavage site within the engineered microcompartment protein. In the method, the at least one second polynucleotide is operably linked to one or more second regulatory elements configured to enable expression in the bacterial cell of the at least one protease. In the method, the introducing is performed to obtain the non-native protein from the engineered microcompartment protein upon cleaving of the at least one first protease cleavage site and/or the at least one second protease cleavage site by the at least one protease expressed in the bacterial cell.
[0037] According to a sixth aspect a system to produce a non-native protein from a bacterial cell comprising at least one engineered microcompartment protein herein described in which the target protein is a protein non-native to the cell, the system comprises
[0038] one or more bacterial cells comprising at least one engineered microcompartment protein herein described assembled with one or more same and/or different microcompartment proteins to form at least one engineered microcompartment within the cell.
[0039] The system also comprises
[0040] at least one second polynucleotide encoding for at least one protease, the at least one second polynucleotide operably linked to one or more second regulatory elements leading to the expression of the at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the bacterial cell to obtain the non-native protein; and
[0041] at least one protease capable of targeting the at least one first protease cleavage site and/or the at least one second protease cleavage site of the engineered microcompartment protein to release the non-native protein from the engineered microcompartment protein in the one or more bacterial cells.
[0042] In the system, the one or more bacterial cells, the at least one second polynucleotide, and the at least one protease are used either simultaneously or sequentially in the methods to provide one or more non-native proteins in from one or more bacterial cell comprising an engineered microcompartment protein herein described.
[0043] In some embodiments, the at least one non-native protein in the engineered microcompartment protein is a non-native toxic protein capable of reacting with a native membrane substrate with a reaction resulting in a cell damage, and the engineered microcompartment protein is provided in the cell to shield cell from toxicity during intracellular production of a toxic non-native protein.
[0044] According to a seventh aspect, a vector is described comprising at least one polynucleotide encoding for an engineered microcompartment protein herein described alone or in combination with regulatory elements in accordance with the disclosure.
[0045] According to an eight aspect, a bacterial cell is described obtained by any one of the methods and/or with any one of the systems of the present disclosure.
[0046] According to a ninth aspect, a composition is described. The composition comprises the engineered microcompartment protein, the engineered microcompartment and/or the bacterial cell herein described.
[0047] Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments in connection with expression, production and/or purification in a bacterial cell of one or more proteins toxic to the bacterial cell or precursor thereof.
[0048] Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments to shield bacteria from toxicity during expression, production and/or purification of non-native toxic protein.
[0049] Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be used in some embodiments in connection with expression, production and/or purification in a bacterial cell of one or more proteins degradable in the bacterial thereof.
[0050] Engineered microcompartment proteins and related engineered microcompartments, vectors, cells compositions methods and systems herein described can be applied in several fields, including basic biology research, applied biology, bio-engineering, bio-energy, medical research, medical diagnostics, therapeutics, bio-fuels, and in additional fields where expression, production and/or purification in a bacterial cell of proteins which are degradable and/or cytotoxic to the bacterial cell and/or their precursors can be used.
[0051] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings, incorporated herein by reference in its entirety and the description below. Other features, objects, and advantages will be apparent from the following description, and the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0052] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with detailed description and the examples, serve to explain the principles and implementations of the disclosure.
[0053] FIG. 1A illustrates a crystal structure of an exemplary encapsulin cage with 60 monomer units (one unit highlighted in red). The left panel shows a view from the outside on the five-fold symmetry axis. One pentamer is highlighted in cyan, with one monomer in red. The right panel shows a view to the inside of the shell, which is cut open in the middle and shown in a surface representation. FIG. 1B shows the T. maritima encapsulin monomer showing the A- and P-domains and the protruding E-loop. The monomer is colored in a rainbow scheme from the N terminus (blue) to the C terminus (red), the domains are named according to the homologous gp5 major capsid protein of the HK97 virus (PDB 1OHG). This figure is adapted from Sutter, M., et al., Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nature Structural & Molecular Biology, 2008. 15(9): p. 939-947.
[0054] FIG. 2 illustrates a BLOSUM62 matrix used for sequence alignment of proteins
[0055] FIG. 3 shows a pairwise alignment of an encapsulin from T. maritima (T. mar, SEQ ID NO: 45) and an encapsulin from M xanthus (M xan, SEQ ID NO: 46).
[0056] FIG. 4A shows the consensus sequence of exemplary encapsulins (SEQ ID NO: 132). FIG. 4B illustrates the segmentation of secondary structures of the encapsulins with regard to the consensus sequence.
[0057] FIG. 5 shows an exemplary encapsulin sequence from T. maritima (SEQ ID: 133).
[0058] FIG. 6 shows sequence alignment of 44 exemplary members from the PF04454 protein family and in particular, GI 122304915 (SEQ ID NO: 86), GI 123527077 (SEQ ID NO: 87), GI 123044215 (SEQ ID NO: 887), GI 123046337 (SEQ ID NO: 89), GI 81373191 (SEQ ID NO: 90), (SEQ ID NO: 91), (SEQ ID NO: 92), (SEQ ID NO: 93), (SEQ ID NO: 94), GI 81833634 (SEQ ID NO: 95), GI 74556699 (SEQ ID NO: 96), GI 81347829 (SEQ ID NO: 97), GI 81344013 (SEQ ID NO: 98), GI 123497228 (SEQ ID NO: 99), GI 501179632 (SEQ ID NO: 100), GI 502293893 (SEQ ID NO: 101), GI 501012501 (SEQ ID NO: 102), GI 506340687 (SEQ ID NO: 103), GI 502591318 (SEQ ID NO: 104), GI 501194893 (SEQ ID NO: 105), GI 490598858 (SEQ ID NO: 106), GI 752720587 (SEQ ID NO: 107), GI 501771872 (SEQ ID NO: 108), GI 501163578 (SEQ ID NO: 109), GI 501434203 (SEQ ID NO: 110), GI 501367709 (SEQ ID NO: 111), GI 505232787 (SEQ ID NO: 112), GI 501923113 (SEQ ID NO: 113), GI 502776253 (SEQ ID NO: 114), GI 502633921 (SEQ ID NO: 115), GI 496662878 (SEQ ID NO: 116), GI 500836508 (SEQ ID NO: 117), GI 501047338 (SEQ ID NO: 118), 4PT2_P (SEQ ID NO: 119), GI 501055150 (SEQ ID NO: 120), GI 501691096 (SEQ ID NO: 121), GI 494995233 (SEQ ID NO: 122), GI 501364857 (SEQ ID NO: 123), GI 502892820 (SEQ ID NO: 124), GI 527109103 (SEQ ID NO: 125), GI 501346422 (SEQ ID NO: 126), GI 501373147 (SEQ ID NO: 127), GI 501587999 (SEQ ID NO: 128), GI 500074236 (SEQ ID NO: 129), GI 501827525 (SEQ ID NO: 130), GI 521295581 (SEQ ID NO: 131)
[0059] FIG. 7 shows a schematic illustration of insertion sites for target proteins, protease cleavage sites and/or tags in embodiments herein described. In particular, FIG. 7A shows insertion sites of a target protein in engineered microcompartment herein described. FIG. 7B shows insertion sites of a target protein in engineered microcompartment herein described. FIG. 7B FIG. 7C and FIG. 7D shows insertion sites of a protease cleavage sites in engineered microcompartment herein described.
[0060] FIG. 8 shows in one embodiment an exemplary engineered microcompartment protein comprising Apidaecin 1a peptide fused to the N-terminus of an encapsulin protein from M xanthus through a TEV protease cleavage site and a linker region (SEQ ID: 134).
[0061] FIGS. 9A-B illustrate two exemplary systems for encapsulation of AMPS.
[0062] FIG. 10 illustrates a design of sense-control-release systems for P. aeruginosa. Panel A) Primary circuit strategy. LasR and AMP-encapsulin are produced from constitutive sigma70 promoters. 3OC12HSL-bound LasR drives expression of an ECF sigma factor (ECFsf), which in turn drives expression of the protease/lysis cassette as well as an anti-sigma factor. The anti-sigma factor inhibits the ECFsf and thus turns off expression of the protease/lysis cassette in a negative feedback loop. Panel B) Alternative circuit where 3OC12HSL-bound LasR drives expression of AMP-encapsulin and the ECFsf.
[0063] FIG. 11 illustrates the design of a protease-sensitive encapsulin.
[0064] FIG. 12 illustrates an exemplary testing of therapeutic delivery system efficacy in bacterial liquid culture.
[0065] FIG. 13 illustrates an exemplary testing of therapeutic delivery system efficacy in biofilm.
[0066] FIG. 14 illustrates an exemplary testing of therapeutic delivery system efficacy in in host tissue culture model
[0067] FIGS. 15A-C illustrate the design and sequences of the exemplary protease-sensitive AMP-encapsulin fusions(SEQ ID NO: 135-138).
[0068] FIG. 16 illustrate exemplary Ap-containing constructs as controls.
[0069] FIG. 17 shows in one embodiment expression of constructs pMCY124, pMCY125 and pMCY133 in comparison with control constructs.
[0070] FIG. 18 shows in one embodiment purification of constructs pMCY124, pMCY125 and pMCY133.
[0071] FIGS. 19A-C show in one embodiment TEV protease cleavage of the purified Ap-encapsulin fusions. All samples were analyzed by SDS-PAGE using both an any-kDa gel to analyze fragment >15 kDa (FIG. 19A) and a 16.5% Tris-Tricine gel to analyze fragments <15 kDa (FIG. 19B). Samples were also analyzed by Western blot using an anti-TEV site antibody (FIG. 19C). Arrows on the gels above show digested fragments.
[0072] FIG. 20A illustrates gene cassettes expressing up to 4 Ap peptides fused to a single Encapsulin construct. FIG. 20B shows the expression of the gene cassettes in FIG. 19A with the samples resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue. Red arrows denote predicted location of the expressed protein.
[0073] FIG. 21A illustrates gene cassettes expressing up to 3 HB peptides fused to a single Encapsulin construct as well as a control of HB fused to thioredoxin (Trx). FIG. 21B shows the expression of the gene cassettes in FIG. 21A with the samples resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue. Red arrows denote predicted location of the expressed protein.
[0074] FIG. 22A shows a schematic illustration of gene constructs for HBCM2 fusions with engineered Enc. TEV denotes TEV protease recognition sites. 6.times. His denotes a hexa-histidine tag for purification. Linkers shown in light gray are described in FIG. 22B. FIG. 22B shows a table reporting the amino acid sequences of HB-Enc constructs (SEQ ID NO: 48-54) and fusions of HB peptide to other common carrier proteins (SEQ ID NO: 55-58). Plain text, indicates Enc or carrier protein sequence, Italics, linker; Bold, TEV recognition site; Bold underlined, HBCM2; Italics underlined, His-tag.
[0075] FIG. 23A shows images illustrating the results of experiments to detect the expression of the HB fusion constructs in FIGS. 22A-B in C43(DE3) E. coli from a T7 IPTG inducible promoter. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue (top) or blotted to a PVDF membrane and probed with mouse anti-His.sub.6 primary antibody and rabbit anti-mouse HRP conjugated secondary antibody (bottom). T denotes the total cell lysate, while S denotes the soluble fraction. Arrows denote expected size of the expressed protein. FIG. 23B shows a chart illustrating the final OD.sub.600 of the C43(DE3) E. coli culture after overnight induction at 18.degree. C.
[0076] FIG. 24 shows an image of a gel including purified HB-Enc fusion proteins with or without TEV protease digestion at 4.degree. C. overnight. Samples were resolved on a 16.5% Tris-Tricine gel and stained with Commassie blue. In the bottom portion a table shows the normalized ratio of HB released/HB-Enc fusion based on densitometry, assuming the HB/HB-Enc ratio for HB-EncK71.sup.TEVK138.sup.TEV-His is 1.
[0077] FIG. 25 shows an image of a gel including purified HB-Enc fusion proteins with a GT linker replacing the flexible G.sub.4T linker between the HB peptide and the N-terminus of Enc. Fusions were digested with or without TEV protease at 4.degree. C. overnight and samples were resolved on a 16.5% Tris-Tricine gel and stained with Commassie blue.
[0078] FIG. 26 shows an image illustrating the results of a Native PAGE analysis of the HB-Enc constructs. Samples were resolved on an any-kDa Native PAGE gel and stained with Coomassie blue. Arrows denote the majority species. High molecular weight (MW) species are located at the top of the gel in the well area. Low MW species enter the gel and migrate lower into the gel.
[0079] FIG. 27 shows charts illustrating size exclusion chromatography traces of absorbance at 280 nm for a cage-forming construct (Ap-EncK138.sup.TEV-His) and a non-cage forming construct (Ap-EncK71.sup.TEVK138.sup.TEV-His). Dashed line denotes the retention time of a 670 kDa standard. The void volume is labeled. Cage-forming, high MW species is denoted by a black arrow, while the non-cage-forming, low MW species is denoted by a white arrow.
[0080] FIG. 28 shows TEM images of cage-forming (EncK138.sup.His, HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEV-His) and non-cage forming (HB-EncK71.sup.TEVK138.sup.TEV-His, HB-EncK71.sup.TEV) HB-Enc constructs. Scale bar is 100 nm.
[0081] FIG. 29 shows an image illustrating Native PAGE analysis and TEM images of Ap-Enc fusions: Ap-EncK138.sup.TEV-His and Ap-EncK71.sup.TEVK138.sup.TEV-His. Black and white arrows denote high and low MW species, respectively. Scale bar on TEM images is 100 nm.
[0082] FIG. 30 shows results of experiments illustrating protease sensitivity of exemplary HB-Enc constructs in cell lysate. C43(DE3) E. coli expressing the constructs were either 1) lysed in the presence of BPER-II and lysozyme; 2) lysed in the absence of BPER-II by French pressure lysis; or 3) lysed in the absence of BPER-II by French pressure lysis and then incubated at 4.degree. C. overnight. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue (top) or blotted to a PVDF membrane and probed with mouse anti-His.sub.6 primary antibody and rabbit anti-mouse HRP conjugated secondary antibody (bottom). Black arrows denote size of full-length fusions. White arrows denote sizes of proteolysis products.
[0083] FIG. 31 Panel A shows anti-bacterial growth inhibition assays against E. coli BL21(DE3) for TEV-digested HB-EncK71.sup.TEVK138.sup.TEV and negative controls of undigested HB-EncK71.sup.TEVK138.sup.TEV alone and TEV protease alone. Only TEV-digested HB-EncK71.sup.TEVK138.sup.TEV has activity. FIG. 31 Panel B shows inhibition assays for various concentrations of chemically synthesized M-HBCM2-TEV peptide. The peptide has an MIC <5 .mu.g/mL, consistent with native HBCM2 peptide.
[0084] FIG. 32 shows charts illustrating the results of anti-bacterial growth inhibition assays against E. coli BL21(DE3) for various concentrations of chemically synthesized native AP peptide (FIG. 32 Panel A) and M-AP-TEV peptide (FIG. 32 Panel B). Data shows M-AP-TEV peptide is not active compared to native AP peptide.
[0085] FIG. 33A shows expression of the HB fusion constructs in FIGS. 22A-B in TOP10 E. coli from a pBAD arabinose inducible promoter. Cells were induced with either 40 .mu.M or 10 mM arabinose. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. T denotes the total cell lysate, while S denotes the soluble fraction. FIG. 33B shows comparison of full-length HB fusions expressed in either C43(DE3) cells (C43) or BL21(DE3) cells (BL21) versus HB fusions expressed in TOP10 cells from a pBAD promoter (Ara). In both FIGS. 33A and B, black arrows denote expected size of the expressed protein, while white arrows denote truncation products. FIG. 33C shows the final OD.sub.600 of the TOP10 E. coli cultures after 4-5 h induction at 37.degree. C.
[0086] FIG. 34 shows results of experiments directed to illustrate expression and protease sensitivity of exemplary constructs HB-Trx, HB-SUMO, HB-GST, and HB-MBP. FIG. 34 Panel A shows an image of a gel illustrating expression of the HB-Trx, HB-SUMO, HB-GST, and HB-MBP constructs in FIGS. 22A-B in BL21(DE3) E. coli from a T7 IPTG inducible promoter. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. T denotes the total cell lysate, while S denotes the soluble fraction. FIG. 34 Panel B shows an image of a gel illustrating protease sensitivity of the constructs in cell lysate. BL21(DE3) E. coli expressing the constructs were either 1) lysed in the presence of BPER-II and lysozyme; 2) lysed in the absence of BPER-II by French pressure lysis; or 3) lysed in the absence of BPER-II by French pressure lysis and then incubated at 4.degree. C. overnight. Samples were resolved on an any-kDa SDS-PAGE gel and stained with Commassie blue. For both Panels A and B, black arrows denote size of full-length fusions.
[0087] FIG. 35 shows an image of a gel illustrating isolation of M-Ap-TEV peptide following TEV protease cleavage of Ap-EncK71.sup.TEVK138.sup.TEV-His. Samples were collected before and after cleavage of Ap-EncK71.sup.TEVK138.sup.TEV-His and peptide was collected in the filtrate after centrifugation on a 10 kDa molecular weight cutoff centrifugal filter. Samples were resolved on a 16.5% Tris-Tricine SDS-PAGE gel and stained with Commassie blue.
DETAILED DESCRIPTION
[0088] Provided herein are engineered microcompartment proteins and related engineered microcompartments, bacterial cells, compositions, methods and systems that can be used in several embodiments for expression, production and/or purification in a bacterial cell of proteins non-native to the bacterial cell.
[0089] The term "microcompartment" or "bacterial microcompartment" as used herein indicated organelles within a bacterial cell in which a protein shell encloses enzymes and other proteins. Microcompartments are typically about 40-200 nanometers in diameter and are entirely made of proteins in which the shell functions like a membrane, as it is selectively permeable. Exemplary microcompartments are described in application Ser. No. 15/178,454 filed on Jun. 6, 2016 and published on Dec. 15, 2016 with publication number US206/0362697 incorporated herein by reference in its entirety.
[0090] In embodiments herein described, the microcompartments are encapsulin microcompartments and the related microcompartment proteins are encapsulins or encapsulin like
[0091] The term "encapsulin" or "encapsulin-like" as used herein indicates proteins that are capable of self-assembling in a bacterial cell to form a microcompartment in which interior molecules (e.g., DNA, RNA, protein) can be encaged. In some instances encapsulin proteins can be native to the bacterial cells where they are expressed.
[0092] Accordingly, the wording "encapsulin-like microcompartments" or "cage" or "BMC" as used herein refers to organelles produced, and in particular possibly natively produced, by bacteria or viruses to organize and sequester biological molecules, such as DNA, RNA, or protein in a bacterial cell within the confines of a protein shell. Accordingly encapsulin microcompartment can be native or non-native to the cells where produced as would be understood by a skilled person. The encapsulin microcompartments typically have pseudo-icosahedral structures that can be 10 to 400 nm in diameter with a thickness of 20-30 .ANG. [1]. Encapsulin-like protein protomers assemble into pentameric and/or hexameric shapes that further assemble to form the icosahedral microcompartment where the pentagons form the vertices and the hexagons form the flat facets of the compartment. All compartments have a total 20 T triangular faces that are formed from 12 pentagons and 10(T-1) hexagons. T is defined as the triangulation number, which can be any non-negative integer that fits T=h 2+k 2+hk, where h and k are also non-negative integers (e.g., T can be 1, 3, 4, 7, etc.). Compartments with different numbers of protomers and T values can result in different sized and shaped compartments.
[0093] Encapsulin microcompartments typically comprise a microcompartment protein or shell protein forming the shell of the microcompartment and one or more interior proteins identifiable by a skilled person. An encapsulin shell protein typically has three common conserved domains: a peripheral domain (P-domain), an axial domain (A-domain), and an elongated loop (E-loop). Common examples of encapsulin shell proteins include encapsulins Enc A from Thermotoga maritima and Myxococcus xanthus, and Pfv from P. furiosus. [1].
[0094] Common examples of encapsulins interior proteins comprise EncB, EncC, and EncD from M xanthus wherein EncB and EncC are ferritin-like proteins that are thought to bind and sequester iron, while EncD has unknown function. Together they are thought to sequester iron under oxidative stress conditions [McHugh, 2014]. Other interior exemplary encapsulins comprise the dye-decoloring peroxidase DyP protein from T maritima as an internal protein.
[0095] Encapsulin-like microcompartments can be of bacterial or viral origin. Exemplary encapsulin-like proteins of bacterial origin include encapsulin or virus-like compartments from Thermotoga maritima (T=1, 24 nm diameter), Pyrococcus furiosus (T=3, 31 nm diameter), and Myxococcus xanthus (T=3, 31 nm diameter). Exemplary encapsulin-like proteins of viral origin include HK97 phage capsid (T=7, 66 nm diameter).
[0096] A representative example of encapsulin proteins is provided by Thermotoga maritima encapsulin. In T. maritima encapsulin microcompartment, sixty monomers of T. maritima encapsulin assemble into a spherical superstructure with icosahedral T=1 symmetry, a diameter of 230-240 .ANG. and a thickness of 20-25 .ANG.. [2] as shown in FIG. 1A. The peripheral domain (P-domain), axial domain (A-domain), and elongated loop (E-loop) of Enc A of T. maritima are schematically shown in FIG. 1B).
[0097] Microcompartments from bacteria can be isolated and detected by methods and systems exemplified herein (see Example 28) and by additional methods and systems identifiable by a skilled person upon review of the present disclosure.
[0098] In embodiments herein described, microcompartment proteins comprised within engineered microcompartment proteins of the disclosure are encapsulin proteins having sequence
[0099] X.sub.1--X.sub.2--X.sub.3--X.sub.4--X.sub.5--X.sub.6--X.sub.7--X.su- b.8--X.sub.9--X.sub.10--X.sub.11--X.sub.12--X.sub.13--X.sub.14--X.sub.15--- X.sub.16--X.sub.17--X.sub.18--X.sub.18--X.sub.20--X.sub.21--X.sub.22--X.su- b.23--X.sub.24--X.sub.25--X.sub.26--X.sub.27--X.sub.28--X.sub.29--X.sub.30 X.sub.31--X.sub.32--X.sub.33 X.sub.34--X.sub.35--X.sub.36--X.sub.37--X.sub.38--X.sub.39--X.sub.40--X.s- ub.41--X.sub.42--X.sub.43--X.sub.44--X.sub.45--X.sub.46--X.sub.47--X48--X.- sub.49--X.sub.50--X51--X.sub.52--X.sub.53--X.sub.54--X.sub.55--X56--X.sub.- 57--X.sub.58--X.sub.59--X.sub.60--X.sub.60--X.sub.61--X.sub.62--X.sub.63--- X.sub.64--X.sub.65--X.sub.66--X.sub.67--X.sub.68--X.sub.69--X.sub.70--X.su- b.71--X.sub.72--X.sub.73--X.sub.74--X.sub.75--X.sub.76--X.sub.77--X.sub.78- --X.sub.79--X.sub.80--X.sub.81--X.sub.82--X.sub.83--X.sub.84--X.sub.85--X.- sub.86--X.sub.88--X.sub.89--X.sub.90--X.sub.91--X.sub.92--X.sub.93--X.sub.- 94--X.sub.95--X.sub.96--X.sub.97--X.sub.98--X.sub.99--X.sub.100--X.sub.101- --X.sub.102--X.sub.103--X.sub.104--X.sub.105--X.sub.106--X.sub.107--X.sub.- 108--X.sub.109--X.sub.110--X.sub.111--X.sub.112--X.sub.113--X.sub.114--X.s- ub.115--X.sub.116--X.sub.117--X.sub.118--X.sub.119--X.sub.120--X.sub.121--- X.sub.122--X.sub.123--X.sub.124--X.sub.125--X.sub.126--X.sub.127--X.sub.12- 8--X.sub.129--X.sub.130--X.sub.131--X.sub.132--X.sub.133--X.sub.134--X.sub- .135--X.sub.136--X.sub.137--X.sub.138--X.sub.139--X.sub.140--X.sub.141--X.- sub.142--X.sub.143--X.sub.144--X.sub.145--X.sub.146--X.sub.147--X.sub.148-- -X.sub.149--X.sub.150--X.sub.151--X.sub.152--X.sub.153--X.sub.154--X.sub.1- 55--X.sub.156--X.sub.157--X.sub.158--X.sub.159--X.sub.160--X.sub.161--X.su- b.162--X.sub.163--X.sub.164--X.sub.165--X.sub.166--X.sub.167--X.sub.168--X- .sub.169--X.sub.170--X.sub.171--X.sub.172X.sub.173--X.sub.174--X.sub.175--- X.sub.176--X.sub.177--X.sub.178--X.sub.179--X.sub.180--X.sub.181--X.sub.18- 2--X.sub.183--X.sub.184--X.sub.185--X.sub.186--X.sub.187--X.sub.188--X.sub- .189--X.sub.190--X.sub.191--X.sub.192--X.sub.193--X.sub.194--X.sub.195--X.- sub.196--X.sub.197--X.sub.198--X.sub.199--X.sub.200--X.sub.201--X.sub.202-- -X.sub.203--X.sub.204--X.sub.205--X.sub.206--X.sub.207--X.sub.208--X.sub.2- 09--X.sub.210--X.sub.211--X.sub.212--X.sub.213--X.sub.214--X.sub.215--X.su- b.216--X.sub.217--X.sub.218--X.sub.219--X.sub.220--X.sub.221--X.sub.222--X- .sub.223--X.sub.224--X.sub.225--X.sub.226--X.sub.227--X.sub.228--X.sub.229- --X.sub.230--X.sub.231--X.sub.232--X.sub.233--X.sub.234--X.sub.235--X.sub.- 236--X.sub.237--X.sub.238--X.sub.239--X.sub.240--X.sub.241--X.sub.242--X.s- ub.243 --X.sub.244--X.sub.245--X.sub.246--X.sub.247--X.sub.248--X.sub.249 X.sub.250--X.sub.251--X.sub.252--X.sub.253 (SEQ ID NO: 1).
[0100] in which X.sub.1 is M, X.sub.2 is D, X.sub.3 is N, X.sub.4 is L, X.sub.5 is K, X.sub.6 is R, X.sub.7 is E, X.sub.8 is L, X.sub.9 is A, X.sub.10 is P, X.sub.11 is L, X.sub.12 is T, X.sub.13 is E, X.sub.14 is E, X.sub.15 is A, X.sub.16 is W, X.sub.17 is A, X.sub.18 is E, X.sub.19 is I, X.sub.20 is D, X.sub.21 is E, X.sub.22 is E, X.sub.23 is A, X.sub.24 is R, X.sub.25 is E, X.sub.26 is T, X.sub.27 is A, X.sub.28 is K, X.sub.29 is R, X.sub.30 is H, X.sub.31 is L, X.sub.32 is A, X.sub.33 is G, X.sub.34 is R, X.sub.35 is R, X.sub.36 1S V, X.sub.37 1S V, X.sub.3g is D, X.sub.39 is V, X.sub.40 is E, X.sub.41 is G, X.sub.42 is P, X.sub.43 is L, X.sub.44 is G, X.sub.45 1S W, X.sub.46 is G, X.sub.47 is Y, X.sub.48 is S, X.sub.49 is A, X.sub.50 is V, X.sub.51 is P, X.sub.52 is L, X.sub.53 is G, X.sub.54 is R, X.sub.55 is L, X.sub.56 is E, X.sub.57 is E, X.sub.58 is I, X.sub.59 is E, X.sub.60 is G, X.sub.61 is P, X.sub.62 is A, X.sub.63 is E, X.sub.64 is G, X.sub.65 is V, X.sub.66 is Q, X.sub.67 is A, X.sub.68 is G, X.sub.69 is V, X.sub.70 is R, X.sub.71 is Q, X.sub.72 is V, X.sub.73 is L, X.sub.74 is P, X.sub.75 is L, X.sub.76 is P, X.sub.77 is E, X.sub.78 is L, X.sub.79 is R, X.sub.g0 is V, X.sub.81 is P, X.sub.82 is F, X.sub.83 is T, X.sub.84 is L, X.sub.85 is S, X.sub.86 is R, X.sub.87 is R, X.sub.88 is D, X.sub.89 is L, X.sub.90 is D, X.sub.91 is A, X.sub.92 is V, X.sub.93 is E, X.sub.94 is R, X.sub.95 is G, X.sub.96 is A, X.sub.97 is K, X.sub.98 is D, X.sub.99 is L, X.sub.100 is D, X.sub.101 is L, X.sub.102 is S, X.sub.103 is P, X.sub.104 is V, X.sub.105 is VA, X.sub.106 is E, X.sub.107 is A, X.sub.108 is A, X.sub.109 is R, X.sub.110 is L, X.sub.111 is L, X.sub.112 is A, X.sub.113 is R, X.sub.114 is A, X.sub.115 is E, X.sub.116 is D, X.sub.117 is R, X.sub.118 is L, X.sub.119 is I, X.sub.120 is F, X.sub.121 is N, X.sub.122 is G, X.sub.123 is Y, X.sub.124 is A, X.sub.125 is E, X.sub.126 is A, X.sub.127 is G, X.sub.128 is I, X.sub.129 is E, X.sub.130 is G, X.sub.131 is L, X.sub.132 is L, X.sub.133 is N, X.sub.134 is A, X.sub.135 1S S, X.sub.136 is G, X.sub.137 is N, .sub.X138 is L, X.sub.139 is K, X.sub.140 is L, X.sub.141 is P, X.sub.142 is L, X.sub.143 is S, X.sub.144 is A, X.sub.145 is D, X.sub.146 is P, X.sub.147 is G, X.sub.148 is D, X.sub.149 is I, X.sub.150 is P, X.sub.151 is D, X.sub.152 is A, X.sub.153 is I, X.sub.154 is A, X.sub.155 is E, X.sub.156 is A, X.sub.157 is L, X.sub.158 is T, X.sub.159 is K, X.sub.160 is L, X.sub.161 is R, X.sub.162 is E, X.sub.163 is A, X.sub.164 is G, X.sub.165 is V, X.sub.166 is E, X.sub.167 is G, X.sub.168 is P, X.sub.169 is Y, X.sub.170 is A, X.sub.171 is L, X.sub.172 is V, X.sub.173 is L, X.sub.174 is S, X.sub.175 is P, X.sub.176 is D, X.sub.177 is L, X.sub.178 is Y, X.sub.179 is T, X.sub.180 is A, X.sub.181 is L, X.sub.182 is F, X.sub.183 is R, X.sub.184 is V, X.sub.185 is Y, X.sub.186 is D, X.sub.187 is G, X.sub.188 is T , X.sub.189is G, X.sub.190 is Y, X.sub.191 is P, X.sub.192 is E, X.sub.193 is I, X.sub.194 is E, X.sub.195 is H, X.sub.196 is I, X.sub.197 is K, X.sub.198 is E, X.sub.199 is L, X.sub.200 is V, X.sub.201 is D, X.sub.202 is G, X.sub.203 is G, X.sub.204 is V, X.sub.205 is I, X.sub.206 is W, X.sub.207 is A, X.sub.208 is P, X.sub.209 is A, X.sub.210 is L, X.sub.211 is D, X.sub.212 is G, X.sub.213 is G, X.sub.214 is A, X.sub.215 1S V, X.sub.216 is L, X.sub.217 1S V, X.sub.218 is S, X.sub.219 is T, X.sub.220 is R, X.sub.221 is G, X.sub.222 is G, X.sub.223 is D, X.sub.224 is F, X.sub.225 is D, X.sub.226 is L, X.sub.227 is T, X.sub.228 is L, X.sub.229 is G, X.sub.230 is Q, X.sub.231 is D, X.sub.232 is L, X.sub.233 is S, X.sub.234 is I, X.sub.235 is G, X.sub.236 is Y, X.sub.237 is L, X.sub.238 is S, X.sub.239 is H, X.sub.240 is D, X.sub.241 is A, X.sub.242 is D, X.sub.243 is N, X.sub.244 is V, X.sub.245 is E, X.sub.246 is L, X.sub.247 is F, X.sub.248 is L, X.sub.249 is T, X.sub.250 is E, X.sub.251 1S S, X.sub.252 is F, X.sub.253 is T (SEQ ID NO: 1) or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:l.
[0101] In preferred embodiments, the engineered microcompartment protein of the disclosure, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 30% sequence identity, a 40% sequence identity, at least a 50% sequence identity, or more preferably at least a 60% sequence identity or greater with SEQ ID NO: 1 or at least 40% sequence similarity, preferably at least 50% sequence similarity, at least 60% sequence similarity or a greater similarity to SEQ ID NO:1.
[0102] The term "sequence identity" indicates the amount of characters that are identical between two different sequences following alignment of the sequences. The percent identity is calculated from the number of exact character matches divided by the total number of aligned characters, including gaps, multiplied by 100%.
[0103] The term "sequence similarity" indicates the amount of characters that are similar between two different sequences following alignment of the sequences. Different from the "identity", which refers to an exact match between two nucleotides or amino acids, similarity refers to a resemblance between two residues that is greater than one would expect at random and provides a predictable structure. Amino acids are considered similar if they have a positive value in a substitution matrix, such as BLOSUM-62 (FIG. 2). BLOSUM-62 is a probability matrix based on observed substitutions found in a broad sampling highly aligned sequences [3] and is used in the BLAST alignment tool. Percent similarity is calculated from the number of similar amino acid matches (based on the substitution matrix) divided by the total number of aligned characters, including gaps, multiplied by 100%.
[0104] Sequence identity and sequence similarity can be detected by commonly used searching programs, like BLAST, PSI-BLAST [4], SSEARCH [5] [6], FASTA [7] and the HMMER3 [8] which can produce accurate statistical estimates of protein sequences that share sequence identity, similarity and also have similar structures.
[0105] The identity or similarity between sequences is typically measured by a process that comprises the steps of aligning two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, characters similar or identical between the two aligned sequences, and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity or identity result is expressed as a percentage.
[0106] An exemplary alignment is illustrated in FIG. 3 which shows a pairwise alignment of encapsulin from T. maritima compared to encapsulin from M xanthus. In the representative example of FIG. 3 a total of 279 residues are aligned. 64 of the residues have an exact match between the two sequences which is denoted as the given letter symbol in the line between the two sequences. In this case, the percent identity is 64/279.times.100%=23% identity. 122 of the residues (including the 64 exact identities) have positive values based on the BLOSUM-62 similarity matrix, which are denoted with either a +symbol or the letter symbol in the line between the two sequences. In this case, the percent similarity is 122/279.times.100%=43%.
[0107] In embodiments herein described, an encapsulin protein which either has SEQ ID NO: 1 or a sequence of the disclosure with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises a P-Domain divided into a first fragment, a second fragment and a third fragment, an E-Loop and an A-Domain, each having an N-terminus and a C-terminus.
[0108] The term the "amino terminus" or "N-terminus" indicate the amino acid residue of a linear polypeptide chain at one of the extremities of the linear polypeptide chain which, when not involved in a peptide bond, presents an amino group. The term the "carboxyl terminus" "C-terminus" indicate the amino acid residue of a linear polypeptide chain at one of the extremities of the linear polypeptide chain which, when not involved in a peptide bond, presents a carboxyl group. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NH2-group). An N terminus or a C-terminus of a polypeptide is typically comprised within a "tail" of the protein which indicates a segment or fragment at the related end of the protein.
[0109] The term "fragment` or "segment" as related to the protein indicates any continuous part of a protein sequence from single amino acid up to the full protein associated to an identifiable structure within the protein. An "identifiable structure" in the sense of the disclosure indicates a spatial arrangement of the primary structure or portions thereof which can be detected by techniques such as crystallography, hydrophobicity analysis or additional techniques known by a skilled person. In some instances, a protein segment or fragment can comprise one or more secondary structures of the protein.
[0110] The "secondary structure" of a protein refers to local sub-structures with a repeating geometry identifiable within crystal structure of the protein, circular dichroism or by additional techniques identifiable by a skilled person. In some instances, a secondary structure of a protein can be identified by the patterns of hydrogen bonds between backbone amino and carboxyl groups. Secondary structures can also be defined based on a regular, repeating, geometry, being constrained to approximate values of the dihedral angles .psi. and .phi. of the amino acids in the secondary structure unit on the Ramachandran plot. Two main types of secondary structure are the alpha helix .alpha.nd the beta strand or beta sheets as will be identifiable by a skilled person. Both the alpha helix and the beta sheet represent a way of establishing non-covalent hydrogen bonds between constituents of the peptide backbone. Secondary structure formation can be promoted by formation of hydrogen bonds between backbone atoms. Amino acids that can minimize formation of a secondary structure by destabilizing the structure of the hydrogen bonding interactions are referred to as secondary structure breakers. Amino acids that can promote formation of a secondary structure by stabilizing formation of hydrogen bonding interactions are referred to as structure makers.
[0111] Several sequential secondary structures may form a "supersecondary unit" or "structural motif." A "supersecondary unit" or "structural motif" indicates a segment of the protein that forms an identifiable three-dimensional structure formed by adjacent secondary structure elements optionally linked by unstructured protein regions. In structural motifs the secondary structures are typically comprised with a same orientation one with respect to another. In particular some structural motifs (e.g. zinc fingers, a Greek key or helix--turn helix) are conserved in different proteins as will be understood by a skilled person.
[0112] The "tertiary structure" of a protein refers to the three-dimensional structure of a protein, stabilized by non-covalent interactions among non-adjacent segments of the protein and optionally by one or more additional compounds or ions interacting through covalent or non-covalent interactions with one or more segments of the proteins. Exemplary non-covalent interactions stabilizing the three dimensional structure of the proteins comprise non-specific hydrophobic interactions, burial of hydrophobic residues from water, specific tertiary interactions, such as salt bridges, hydrogen bonds, the tight packing of side chains, chelation and disulfide bonds and additional interactions identifiable by a skilled person. Exemplary covalent interactions among compounds or ions and segments of the protein comprise, N-linked glycosylation, cytochrome C heme attachment and additional interaction identifiable by a skilled person.
[0113] In embodiments herein described, the first fragment of the P-Domain of an encapsulin protein of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus C-terminus direction, a 4 to 26 residues alpha helix .alpha.l, followed by a 0 to 22 residues first non-structured region (e.g. forming a loop), linked to a 4 to 11 residues alpha helix .alpha.2, linked to a 3 to 9 residues beta strand .beta.1, linked to a 3 to 13 residues second non-structured region. In particular, in embodiments, where the encapsulin protein has SEQ ID NO: 1, the first fragment of the P-Domain can be formed by residues X2 to X46. A representative example of the structure of a first fragment of the P-domain according to these embodiments is provided by the first segment of the P-domain of the encapsulin shell protein EnCA illustrated in FIG. 1B. As shown, the first segment of the P-Domain in the representative EncA of FIG. 1B consists of a mixed .alpha./.beta. structure, contains the N terminus and is fragmented with regard to primary sequence (FIG. 1B, orange secondary structure bars). The first fragment or segment of the P-domains contains two alpha helices .alpha.1, .alpha.2 and one beta strand .beta.1.
[0114] In embodiments herein described, the second fragment of the P-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus to C-terminus direction a 9 to 15 residues beta strand .beta.4, linked to a 6 to 15 residues alpha helix .alpha.3, linked to a 0 to 10 residues first non-structured region, linked to a 18 to 29 residues alpha helix .alpha.4, linked to a 9 to 21 residues second non-structured region. In particular, in embodiments, where the encapsulin protein has SEQ ID NO: 1, the second fragment of the P-Domain can be formed by residues X75 to X130. A representative example of the structure of a second fragment of the P-domain according to these embodiments is provided by the second segment of the P-domain of the encapsulin shell protein EnCA illustrated in FIG. 1B. As shown, the second segment of the P-Domain in the representative EncA of FIG. 1B consists of the second segment of the P-domain contains one beta strand .beta.4 and two alpha helices .alpha.3 and .alpha.4.
[0115] In embodiments herein described, the third fragment of the P-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in a N-terminus to C-terminus direction, a 4 to 10 residues beta strand .beta.9, linked to by a 3 to 16 residues first non-structured region, linked to a 7 to 13 residues beta strand .beta.10, linked to a 1 to 15 residues second non-structured region, linked to a 10 to 19 residues beta strand .beta.11. In particular, embodiments, where the microcompartment protein has SEQ ID NO: 1, the third fragment of the P-Domain can be formed by residues X221 to X253. A representative example of the structure of a second fragment of the P-domain according to these embodiments is provided by the third segment of the P-domain of the encapsulin shell protein EncA illustrated in FIG. 1B. As shown, the third segment of the P-Domain in the representative EncA of FIG. 1B consists of the third segment of the P-domain contains three beta strands .beta.9, .beta.10, and .beta.11. A conserved hydrophobic core is located between the helical and .beta.-sheet regions.
[0116] In the engineered microcompartment protein, the E-Loop of the encapsulin protein of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form in a folded encapsulin protein, a secondary structure comprising in an N-terminus to C-terminus direction, a 8 to 16 residues beta strand .beta.2, linked to a 2 to 24 residues first non-structured region, linked to a 7 to 15 residues beta strand .beta.3, linked to a 0 to 6 residues second non-structured region. In particular, in embodiments, where the microcompartment protein has SEQ ID NO: 1, the E-Loop can be formed by residues X47 to X74. A representative example of the structure of an E-Loops according to these embodiments is provided by the E-Loop of the encapsulin shell protein EnCA is illustrated in FIG. 1B. In the illustration of FIG. 1B, the E-loop adopts a flexible loop conformation and is responsible for the formation of contacts between the two-fold symmetry-related subunits by providing a strand that completes a .beta.-sheet formed by both subunits. The E-loop contains two .beta. strands: .beta.2 and .beta.3.
[0117] In the engineered microcompartment protein, the A-Domain of an encapsulin protein comprising residues of SEQ ID NO: 1 or of a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1, comprises residues configured to form, in a folded encapsulin protein, a secondary structure comprising in an N-terminus to C-terminus direction a 0 to 8 residues beta strand .beta.5, linked to a 1 to 15 residues first non-structured region, linked to a 16 to 23 residues alpha helix .alpha.5, linked to a 3 to 11 residues second non-structured region, linked to a 3 to 11 residues beta strand .beta.6, a 9 to 16 residues alpha helix .alpha.6, a 1 to 24 third non-structured region, linked to a 0 to 16 residues alpha helix .alpha.7, linked to a 0 to 8 residues fourth non-structured region, linked to a 1 to 10 residues beta strand .beta.7, linked to a 1 to 12 residues fifth non-structured region, linked to a 3 to 10 residues beta strand .beta.8, linked to a 2 to 12 residues sixth non-structured region In in particular, in embodiments, where the microcompartment protein has SEQ ID NO: 1, the A-Domain can be formed by residues X131 to X220. A representative example of the structure of an A-Domain according to these embodiments provided by the A-Domain of the encapsulin shell protein EnCA is illustrated in FIG. 1B. In the illustration of FIG. 1B, the A-domain forms a compact structure consisting of three helical segments and a five-stranded .beta.-sheet. The A-domain also contains the C terminus of the encapsulin shell protein. This domain has few connections to the rest of the monomer and mediates the contacts of the five-fold symmetry interface. The A-domain contains four beta strands .beta.5, .beta.6, .beta.7, .beta.8 and three alpha helices .alpha.5, .alpha.6, .alpha.7.
[0118] In an encapsulin of an engineered microcompartment protein herein described, each P-domain, A-domain and E-loop of the engineered microcompartment proteins of the current disclosure has a N-terminus and a C-terminus, and the P-domain, A-domain and E-loop are arranged together in a configuration comprising in a direction N-terminus to C-terminus the first fragment of the P-domain linked to the E-loop linked to the second fragment of the P-domain linked to the A-domain linked to the third fragment of the P-domain. In particular, in encapsulin protein used for constructing engineered microcompartment proteins herein described the C-terminus of the first fragment of P-domain is covalently attached to the N-terminus of the E-loop, the C-terminus of the E-loop is covalently linked to the N-terminus of the second fragment of the P-domain, the C-terminus of the second fragment of the P-domain is covalently attached to the N-terminus of the A-domain, and the C-terminus of the A-domain is covalently attached to the N-terminus of the third fragment of the P-domain (see configuration of the representative EncA of FIG. 1B).
[0119] In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X2 to X46, X75 to X130 and X221 to X253 form a P-domain, in which residues X2 to X46 form a first fragment of the P-domain, X75 to X130 form a second fragment of the P-domain and X221 to X253 form a third fragment of the P-domain.
[0120] In particular, in some embodiments, one insertion site can be located within the loop region of the E-loop between .beta.2 and .beta.3, comprising X57 to X65, another insertion site can be located within .beta.3 of the E-loop, comprising X66 to X74, and/or another insertion site can be located in the flexible region between the N-terminus of the A-domain and .alpha.5, including .beta.3, comprising X132 to X144, as will be understood by a skilled person.
[0121] In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X47 to X74 form an E-loop.
[0122] In some embodiments, the encapsulin protein has SEQ ID NO: 1 or a sequence with at least 22% sequence identity or at least 40% sequence similarity to SEQ ID NO:1 and residues X131 to X220 form an A-domain, residues X221 to X253 form a third fragment of the P-domain
[0123] Various embodiments of encapsulin proteins in the sense of the disclosure that can be used for constructing an engineered microcompartment herein described, have a consensus sequence of SEQ ID NO:2 reported below. The amino acids highlighted in bold in SEQ ID NO: 2 and in other sequences herein described (see e.g. T. maritima encapsulin sequence (FIG. 5) indicates either identical or highly conserved among the different encapsulins used for constructing an engineered microcompartment herein described, unless otherwise indicated. The wording "highly conserved" indicates identical/conserved amino acids passing a 3.0 bit conservation setting based on pfam alignment tool for all 44 sequences in pfam 04454 (see https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi at the filing date of the present disclosure).
TABLE-US-00001 (SEQ ID NO: 2) MDNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WGYSAVPLGRL 55 EEIEGPAEGVQ AGVRQVLPLPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK 110 LARAEDRLIFN GYAEAGIEGLL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV 165 EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR 220 GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT
[0124] FIG. 4 shows an additional representation of consensus sequences SEQ ID NO: 2 with complete indication of the related P-Domain, E-Loop and E Domain (FIG. 4A) as well as a schematic representation of the related configuration in the encapsulin sequence (FIG. 4B).
[0125] In particular, consensus sequence SEQ ID NO: 2 contains 253 amino acids forming a 3-D configuration having a P-domain, A-domain and an E-loop. In particular, in consensus sequence SEQ ID NO: 2, residues DNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WG (SEQ ID NO:3) provide the consensus for the first fragment of the P-domain containing two alpha helices .alpha.1, .alpha.2 and one beta strand .beta.1, in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of the fragment. Residues YSAVPLGRL EEIEGPAEGVQ AGVRQVLP (SEQ ID NO: 4) provide the consensus for the E-loop containing two .beta. strands: .beta.2 and .beta.3. Residues LPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK LARAEDRLIFN GYAEAGIEG (SEQ ID NO:5) provide the consensus for the second fragment of the P-domain containing one beta strand .beta.4 and two alpha helices .alpha.3 and .alpha.4 in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of the fragment. Residues LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6) provide the consensus for the A-domain containing four beta strands .beta.5, .beta.6, .beta.7, .beta.8 and three alpha helices .alpha.5, .alpha.6, .alpha.7 and residues GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT (SEQ ID NO:7) form the third fragment of the P-domain containing three beta strands .beta.9, .beta.10, and .beta.11 in which the amino acids highlighted in bold are either identical or highly conserved among the different encapsulins forming the consensus of each respective fragment.
[0126] A skilled person will be able to identify encapsulin proteins in the sense of the disclosure from the consensus sequence SEQ ID NO: 2 following one or more sequence alignments upon reading of the present disclosure.
[0127] In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described have at least a 30% sequence identity, at least a 40% sequence identity, at least a 50% sequence identity, or at least a 60% sequence identity or greater with the encapsulin protein of SEQ ID NO: 2.
[0128] In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described have at least a 50% sequence similarity, at least a 60% sequence similarity or a greater similarity with the encapsulin shell protein of SEQ ID NO: 2.
[0129] In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the E-Loop which following sequence alignment provide a consensus sequence YSAVPLGRL EEIEGPAEGVQ AGVRQVLP (SEQ ID NO:4) In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise an E-loop having a primary sequence of at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 4. In some of these embodiments the E loop sequences of engineered microcompartment proteins herein described comprise a V57 residue and/or a D60 residue such as 3KDT (T. maritima) YAAHPLGEVEVLSDENEVVKWGLRKSLP (SEQ ID NO: 59), GI 501012501 YTVVPEGRLKKIEDNPGNVCTGMYQVKP (SEQ ID NO: 60), GI 502591318 YAAVNTGELRPIDDTPEDVDMKLRQVQP (SEQ ID NO: 61), GI 501771872 YAAVNTGRRTALEDKAEGASIFQRQVLP (SEQ ID NO: 62), and GI 501367709 FSALGTGHVSRVAADTPGVEALQRHVVR (SEQ ID NO: 63).
[0130] In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the A-domain which following sequence alignment provide a consensus sequence LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6). In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise A-domain having a primary sequence of at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 6. In some of these embodiments the A-domain sequences of engineered microcompartment proteins herein described comprise 3DKT: LLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGH YPLEKRVEECLRGGKIITTPRIEDALVVSER (SEQ ID NO: 64), GI 501179632: LREGTSNPKLALPSSASDYPAAIAAALNQLRLAGVNGPYAVVLGAGVYTALSGG DDEGYPVFRHIESLIDGKIVWAPAIEGGFVLSTR (SEQ ID NO: 65), GI 490598858: LLTEDGIVKFPISNWSEGENPFKDISIGLAKFIENGIVGRKALVVSPNLFVQLQRIQ PGTGTTEYDRINKLLDGNIFSTPVLKDDKAVLVCSE(SEQ ID NO: 66), GI 501923113 ILNAEGAQKLQISDWGQGENPYTDIVKAINMIREKGIVGRFVLCLSQSLYFDLQRI QQGTGMTEAQRISSMIGNLYNVPVIKGKKAALICAE (SEQ ID NO: 67), and GI 496662878: LLTVKGSSKIKKSDWSQGENSFADITAGVAQLAKTGYLGRYALVVSPDLFLDLQ RLQPNTGLLEIDRIKKLIGDNVYMTSVMGPGKAVLVCAE (SEQ ID NO: 68).
[0131] In some embodiments, encapsulin proteins used for constructing an engineered microcompartment herein described, have a sequence of the P-domain which following sequence alignment provides a consensus sequence MDNLKRELAPL TEEAWAEIDEE ARETAKRHLAG RRVVDVEGPLG WG (SEQ ID NO:3) for the first fragment, the consensus sequence LPE LRVPFTLSRRD LDAVERGAKDL DLSPVAEAARK LARAEDRLIFN GYAEAGIEG (SEQ ID NO:5) for the second fragment and the consensus sequence GGDFDLTLGQD LSIGYLSHDAD NVELFLTESFT (SEQ ID NO:7) for the third fragment. In some of those embodiments, the encapsulin proteins in the sense of the disclosure comprise P-domain having a primary sequence with at least 22% sequence identity, 30% sequence identity, a 40% sequence identity, a 50% sequence identity, a 60% sequence identity or greater with respect to SEQ ID NO: 3, 5- and 7.
[0132] An exemplary encapsulin shell protein from T. maritima has a SEQ ID NO: 47 shown in FIG. 5, comprising the P-domain, E-loop and A-domain.
[0133] In some embodiments, the encapsulin proteins herein used for constructing the engineered microcompartment proteins comprise the encapsulin proteins from the protein family PF04454 (Linocin_M18, Encapsulating protein for peroxidase), COG1659, and the DUF2184 superfamily. Proteins in this family are found in eubacteria and archaea, and can form nanocompartments within the bacterium which contain ferritin-like proteins or peroxidases, enzymes involved in oxidative-stress response. Detailed information about this protein family can be found in the pfam website as will be understood by a person skilled in the art (see the website http://pfam.xfam.org/family/PF04454 at the filing date of the present disclosure). A sequence alignment of 44 exemplary members from the PF04454 family are shown in FIG. 6.
[0134] In some embodiments, the encapsulin proteins herein used for constructing the engineered microcompartment proteins comprise the members of Phage capsid pfam05065 (see the website http://pfam.xfam.org/family/PF05065 at the filing date of the present disclosure) and HK97 family of viral capsid proteins (TIGRO1554) that have at least 22% identity or at least 40% similarity to SEQ ID NO:1.
[0135] In some embodiments, encapsulins herein described comprise homologous proteins of the encapsulin protein or SEQ ID NO: 1 or SEQ ID NO: 47 with at least 22% sequence identity or 40% sequence similarity, in which one or more residues forming the P-domain, E-loop and A-domain are replaced with a functionally equivalent residue.
[0136] A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical characteristics include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine and lysine are considered as functionally equivalent residues to histidine.
[0137] In embodiments according to the instant disclosure, a microcompartment protein is engineered to introduce in the encapsulin protein a target protein and at least one protease cleavage site configured to allow release of the target protein from the engineered microcompartment protein upon cleavage of the at least one protease cleavage site in the engineered microcompartment protein.
[0138] In particular in embodiments herein described:
[0139] a target protein having an N-terminus, a C-terminus and a sequence up to 80 amino acids in length is inserted at the N-terminus of the first segment of the P-domain of the encapsulin protein alone or together with a linker and/or a tag;
[0140] at least one first protease cleavage site is inserted between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein alone or together with a linker and/or a tag; and at least one second protease cleavage site is inserted, alone or together with a linker and/or a tag, within 9 to 17 amino acids and/or 0-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein to enable digestion of the encapsulin and release of the target protein.
[0141] As used herein, in relation to proteins, the term "insertion" of a first protein or fragment thereof in a second protein or fragment thereof refers to the introduction of the first protein or fragment thereof in between two adjacent amino acids of the first protein or fragment thereof. As a result, an inserted first protein is located in between the two adjacent amino acids of the second protein.
[0142] In particular, an insertion of a first protein in a second protein is performed by forming a first covalent bond between the N-terminal amino acid of the first protein with a first amino acid of the two adjacent amino acids the second protein, and a second covalent bond between the C-terminal amino acid of the first protein with a second amino acid of the two adjacent amino acids of the second protein. As would be understood by a skilled person, a covalent bond between two amino acids in a protein is typically a peptide bond, which is a covalent bond between a carboxyl group and an amino group of two molecules or portions thereof, which results in releasing a molecule of water.
[0143] Accordingly, an insertion of a second protein in a first protein when performed at a protein level typically results in breaking the peptide bond between the two adjacent amino acids of the first protein and forming two new peptide bonds: one between one of the two adjacent amino acids of the first protein and the N-terminal amino acid of the second protein and the other peptide bond formed between the other one of the two adjacent amino acid of the first protein and the C-terminal amino acid of the second protein.
[0144] In embodiments of the disclosure, insertion at the N-terminus of the first segment of the P-Domain is performed at the position between the N-terminus of the first segment of the P-Domain and the adjacent residue upstream in the encapsulin protein. For example, in embodiments where the encapsulin protein has SEQ ID NO: 1, the non-native protein can be introduced at an insertion position -1 relative to the N-terminus of the first segment of the P-Domain of the encapsulin protein. As a consequence, in embodiments where the encapsulin protein has SEQ ID NO: 1 the target protein or an insertion region comprising the target protein optionally together with a tag and/or a linker can be inserted between the adjacent residues X1 and X2 of the microcompartment protein. A schematic illustration of such insertion is illustrated in FIG. 7A. In some embodiments, the insertion region comprising the target protein alone or together with at least one tag and/or one linker comprises up to 80 amino acid residues.
[0145] In embodiments of the disclosure, insertion of the at least one first protease cleavage site between the C-terminus of the target protein and the N-terminus of the first segment of the P-Domain of the encapsulin protein is performed at the position between the N-terminus of the first segment of the P-Domain and the adjacent residue upstream in the target protein. For example, in embodiments where the encapsulin protein has SEQ ID NO: 1, the target protein can be introduced at an insertion position -1 to position relative to the N-terminus of the first segment of the P-Domain of the encapsulin protein. As a consequence in embodiments where the encapsulin protein has SEQ ID NO: 1 the at least one first protease or an insertion region comprising the at least one first protease optionally together with a tag and/or a linker can be inserted between the residues X2 of the microcompartment protein and the residue immediately upstream residue X2. A schematic illustration of such insertion is illustrated in FIG. 7B in which the residues of the inserted target proteins are indicated as AA. In some embodiments, the insertion region comprising the at least one first protease alone or together with at least one tag and/or one linker, comprises up to 22 amino acid residues.
[0146] In embodiments of the disclosure, insertion of the at least one second protease cleavage site within 9-17 and/or 0-8 amino acids adjacent to the C-terminus of the E-loop of the encapsulin protein and/or within 2-14 amino acids adjacent to the N-terminus of the A-domain of the encapsulin protein, can be performed at any one of the 9 to 17 amino acids and/or 0 to 8 adjacent residues upstream of the C-terminus of the E-Loop and/or 2-14 amino acids downstream of the N-terminus of the A-Domain.
[0147] As a consequence in embodiments where the encapsulin protein has SEQ ID NO: 1 the at least one second protease or an insertion region comprising the at least one second protease optionally together with a tag and/or a linker, can be inserted between any one of residues X57 and X74 of the microcompartment protein and/or between any one of residues X132 to X144 of the microcompartment protein. In particular, in SEQ ID NO: 1 residues X57 to X65 define the loop region of the E-loop domain (the first unstructured region of the E-Loop between beta-strands 132 and 133 of the E-Loop of the protein and residues X66 to X74 define beta strand .beta.3 of the E-loop, and residues X132 to X144 define within the beta-strand .beta.5 and the subsequent second unstructured region of the A Domain of the protein as will be understood by a skilled person.
[0148] A schematic illustration of such insertion is illustrated in FIG. 7C and FIG. 7D. In some embodiments, the insertion regions comprising the at least one second protease alone or together with at least one tag and/or one linker, can independently comprise up to 22 amino acid residues.
[0149] Exemplary sequences showing possible insertion points for the at least one second protease with bolded and bolded italics fonts are YSAVPLGRL EEIEGPAEGV (SEQ ID NO. 4), and LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR (SEQ ID NO: 6).
[0150] Preferred insertion points for the at least one second protease cleavage site in SEQ ID NO: 1 are X.sub.57 (corresponding to V57 in constructs exemplified in the example section of the present disclosure); X.sub.60 (corresponding to D60 in constructs exemplified in the example section of the present disclosure); X.sub.71 (corresponding to K71 in constructs exemplified in the example section of the present disclosure); and X.sub.139 (corresponding to K138 in constructs exemplified in the example section of the present disclosure) which can be provided alone or in any combination selected in view of the resulting engineered microcompartment protein as will be understood by a skilled person upon reading of the present disclosure.
[0151] Exemplary sequences of engineered microcompartment proteins enclosing the above insertion points for the at least one second protease cleavage site are reported below wherein the related residues are reported in bold fonts.
TABLE-US-00002 (SEQ ID NO: 4) YSAVPLGRL EEIEGPAEGVQ AGVRQVLP (SEQ ID NO: 6) LL NASGnLKLPLS ADPGDIPDAIA EALTKLREAGV EGPYALVLSPD LYTALFRVYDG tGYPEIEHIKE LVDGGVIWAPA LDGgAVLVSTR
[0152] In embodiments herein described the engineered microcompartment proteins can be cage forming or non-cage forming depending on the positioning of the at least one second protease cleavage site.
[0153] The wording "cage forming" as used herein indicates an engineered microcompartment protein configured to form upon translation within cytoplasm of a cell or in a cell free environment, an encapsulin like microcompartment as described herein. Conversely, the wording non-cage forming as used herein indicates an engineered microcompartment protein configured not to form upon translation within a cytoplasm of a cell or in a cell free environment, an encapsulin like microcompartment as described herein
[0154] In particular in some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site can be within the loop region of the E-loop domain (the first unstructured region of the E-Loop, between beta-strands .beta.2 and .beta.3 of the protein) to provide a cage forming engineered microcompartment protein. Examples are insertions at any of positions X57 and X65 in SEQ ID NO 1, and any of the position 11 (E) to 19 (V) (EIEGPAEGV- SEQ ID NO: 139) in SEQ ID NO; 4. Representative examples are provided by insertions at residues V57 and D60 in T. maritima encapsulin (see Examples 17 to 24). In particular, in order to obtain cage forming microcompartment proteins one insertion can be performed at any one of the residues (see e.g. the insertion of a GG-ENLYFQG-GG SEQ ID NO: 140 or residue of a same or smaller dimension after 1 residue in the region of the Enc from T. maritima which already has 9 amino acids).
[0155] In some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site on the .beta.5 beta strand prior to .alpha.5 of the A-Domain to provide a cage forming engineered microcompartment protein. Examples are insertions at any of positions X132 and X144 in SEQ ID NO 1, and any of the position 2 (L) to 14 (A) of sequence LNASGnLKLPLSA (SEQ ID NO: 141) in SEQ ID NO: 6.
[0156] In some embodiments, an engineered microcompartment protein can be designed to include the at least one second protease cleavage site in the loop region of the E-loop (in the first unstructured region of the E-loop between .beta.2 and .beta.3) and in the A domain and the resulting engineered microcompartment proteins are also expected to be cage forming.
[0157] In some embodiments, an engineered microcompartment protein can be designed to include at least one second protease cleavage site in the E-loop domain within the beta-strand .beta.3 to provide a non-cage forming engineered microcompartment protein. In particular, a disruption of cage-formation occurs if there is an insertion within .beta.3 of the E-loop (e.g. X71 in SEQ ID NO; 1 and K71 construct in the examples) while cage formation would not be disrupted if there is an insertion after X139 in SEQ ID NO; 1 (see K138 constructs in the examples). In some of these embodiments, the addition of this site can improve the kinetics of peptide release. An example is an insertion at any of positions X66 and X74 in SEQ ID NO 1, and any of the position 20 (Q) to 28 (P) (Q AGVRQVLP SEQ ID NO: 142) in SEQ ID NO; 4. A representative example is provided by insertion following residue K71 in T. maritima encapsulin. (see Examples 17 to 24).
[0158] Non-cage forming insertion point for the at least one second protease are expected to be dominant with respect to the cage formation of engineered microcompartment proteins of the present disclosure.
[0159] Accordingly, engineered microcompartment proteins comprising the at least one protease cleavage site in the .beta.3 strand of the E Loop are expected to be non cage forming when in combination with additional second protease cleavage sites in the A Domain are and/or other regions of the E-Loop also expected to be non cage forming. In particular, any insertion within .beta.3 strand of the E-loop domain is expected to be non-cage forming. As a consequence engineered microcompartment proteins comprising the at least one protease cleavage site in the .beta.3 strand of the E Loop and in the loop region of the E-loop (in the first unstructured region of the E-loop between .beta.2 and .beta.3) are also expected to be non cage forming.
[0160] In some embodiments the at least one first protease cleavage site and the at least one second protease cleavage site comprise a same protease cleavage sites. In some embodiments the at least one first protease cleavage site and the at least one second protease cleavage site comprise different protease cleavage sites.
[0161] The wording "protease cleavage site in the sense of the disclosure indicates target sites for proteolytic cleavage by enzymes such peptidases, proteases or proteolytic cleavage enzymes which break peptide bond between amino acids in proteins. The general nomenclature of cleavage site positions of the substrate were formulated by Schechter and Berger, 1967 [9]and Schechter and Berger, 1968 [10] Accordingly, the cleavage site is designated between P1-P1', incrementing the numbering in the N-terminal direction of the cleaved peptide bond (P2, P3, P4, etc. . . . ). On the carboxyl side of the cleavage site the numbering is incremented in the same way (P1', P2', P3' etc.).
[0162] Protease cleavage sites that can be inserted in engineered microcompartment proteins of the disclosure comprise regions up to 25 residues. In particular, protease cleavage sites are inserted in a configuration which makes them surface accessible. In some embodiments protease cleavage site are included in an unstructured segment or within an alpha helical or beta sheet secondary structured segment. Exemplary protease cleavage sites that can be inserted in engineered microcompartment proteins herein described comprise TEV protease cleavage sites with sequence ENLYFQG, (SEQ ID NO:69) which is unstructured and others identifiable by a skilled person upon reading of the present disclosure (see also Table 2 and Example 3).
[0163] In some embodiments of the engineered microcompartment protein herein described, the at least one cleavage site is comprised within an inserted region of up to 25 residues further comprising linkers and/or tags as will be understood by a skilled person upon reading of the present disclosure.
[0164] In embodiments herein described target proteins that can be inserted comprise any protein having 1 to 80 residues possibly comprised within an inserted region of up to 80 residues further comprising linkers and/or tags as will be understood by a skilled person upon reading of the present disclosure.
[0165] The term "protein" as used herein indicates a polypeptide with secondary, tertiary, and possibly quaternary structure. The protein's secondary, tertiary, and quaternary structure can occur on a variety of length scales (tenths of A to nm) and time scales (ns to s), so that in various instances the secondary, tertiary and possibly quaternary structures are dynamic and not perfectly rigid.
[0166] The term "polypeptide" as used herein indicates a polymer composed of two or more amino acid monomers and/or analogs thereof wherein the portion formed by the alpha carbon, the amine group and the carboxyl group of the amino acids in the polymer forms the backbone of the polymer. As used herein the term "amino acid", "amino acid monomer", or "amino acid residue" refers to any of the naturally occurring amino acids, any non-naturally occurring amino acids, and any artificial amino acids, including both D and L optical isomers of all amino acid subsets. In particular, amino acid refers to organic compounds composed of amine (--NH2) and carboxylic acid (--COOH), and a side-chain specific to each amino acid connected to an alpha carbon. Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity, and pKa. Amino acids can be covalently linked to forma polymer through peptide bonds by reactions between the amine group of a first amino acid and the carboxylic acid group of a second amino acid.
[0167] The term "polypeptide" includes amino acid polymers of any length including full length proteins, as well as analogs and fragments thereof. The polypeptide provides the primary structure of a protein wherein the term "primary structure" of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein "sequence" indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of amino acid residues covalently linked by peptide bond.
[0168] In particular, in some embodiments of the present disclosure, the target protein of the engineered microcompartment protein of the disclosure can be a protein which is non-native to the bacterial cell where the engineered microcompartment protein is configured to be expressed based on the experimental design.
[0169] The wording "native" as used herein with reference to a compound and a cell, identifies a compound, molecule or structure naturally provided and in particular produced in the cell. Therefore, a native protein or a native substrate when described in connection with a cell, refers to a protein and/or substrate that is itself naturally provided and in particular, produced in the referenced cell. Conversely, the term "non-native" as used herein with reference to a protein and/or a substrate in connection with a cell, refers to a protein and/or substrate that is itself not naturally produced in the referenced cell.
[0170] In embodiments wherein the target protein is a non-native protein, the non-native protein herein described include toxic non-native proteins and non-toxic non-native proteins which in some cases can be degraded within a target bacterial cell where expression production and/or purification of the non-native protein is desired. Accordingly, engineered microcompartment proteins compositions, in some embodiments are configured to allow compartmentalization of certain toxic or non-toxic proteins in cells where said proteins are non-native, thus shielding the cell from toxicity from said toxic non-native proteins or shielding the non-native proteins from the cell environment.
[0171] Exemplary non-toxic non-native proteins that can be introduced as target protein in the engineered microcompartment proteins of the disclosure include proteins that can be proteolyzed by protease within the host cell and in in particular, proteins which are particularly sensitive to native proteases present in the host cells. In such cases, the engineered microcompartment proteins can protein the non-native proteins from proteolysis in the host. Exemplary non-native non-toxic proteins include proteins susceptible to Lon protease, OmpT and ClpXP in E. coli. Non-toxic non-native proteins also include proteins that are difficult to fold, including those that require disulfide bonds for proper folding and function. The engineered microcompartment proteins can provide an enclosed structure to prevent aggregation and facilitate proper folding.
[0172] The term "toxic non-native protein", as used throughout, refers to a protein or peptide that is itself not naturally produced by a reference cell and is toxic to the host cell when provided or produced in said cell.
[0173] In particular, toxic non-native proteins in the sense of the disclosure in connection with cells where they are expressed produced and/or purified indicates proteins or peptides that are not native to said cell and can react with a native cellular target substrate to provide cell damage by triggering a series of linked biological or chemical reactions within the cell resulting in damage to said cell.
[0174] The wording "native cellular target substrate" or "native cellular substrate" as used herein indicates a compound molecule or structure that is naturally occurring in a cell and is a part of reactions taking place in the cell to keep the cell alive. Exemplary native cellular target substrates in the sense of the disclosure comprise native cellular lipids, proteins, nucleic acids and/or related cellular structures, such as cell membrane or cell chromatin.
[0175] Exemplary reactions between a native cellular target substrate and a non-native protein, particularly a toxic non-native protein, which exemplary reactions trigger a series of linked biological or chemical reactions in the cell resulting in a damage to the cell, comprise binding and/or bond cleavage resulting in disruption and/or inactivation of the cellular target substrate. For example, targeting of membrane lipids damages the cell membrane which, on its turn, impacts the state of cell electrolytes, e.g. calcium, which when constantly increased, induces apoptosis.
[0176] The word "damage" as used herein refers to a physical harm caused to a cell in such a way as to impair its normal function. In particular, cell damage can occur as a result of disruption the normal homeostasis of an affected cell. Among other causes, cell damage can be due to physical, chemical, or, biological, factors resulting from targeting of cell components such as DNA and the cell membrane. Cell damage can be reversible or irreversible. Depending on the extent of injury, the cellular response may be adaptive and where possible, homeostasis is restored. Cell death occurs when the severity of the injury exceeds the cell's ability to repair itself and can occur by necrosis or apoptosis.
[0177] Toxicity in the sense of the disclosure in particular occurs when a non-native protein interferes with the normal proliferation and homeostasis of the microorganism and the visible result is slower growth rate, low final cell density, and death ([11]-[12]) Toxicity of a non-native protein can therefore be detected with reference cell growth before production of a non-native toxic protein (basal growth) and after detection of possible toxicity of vectors or other expression system for production of the non-native protein within a cell which can be performed with approaches discussed for example in reference (2) or otherwise identifiable by a skilled person upon reading of the present disclosure. After control of basal growth and of toxicity of the expression system, the culture can be grown until the expression of the non-native protein. Following expression of the non-native protein, if the non-native protein is toxic, cell growth will be impaired or arrested depending on the level of toxicity. In some cases, the level of toxicity of a non-native protein can be dependent on a threshold of host tolerance. In such situations, toxicity of a non-native protein can be dependent on the level of expression of the non-native protein in comparison with the threshold of host tolerance which should be reached and exceeded for the protein to have toxicity as will be understood by a skilled person.
[0178] Examples of proteins or peptides that are toxic and therefore harmful to a cell include antimicrobial peptides, as well as proteases and lysins, which are harmful to bacterial cells through direct targeting of cytoplasmic, membrane, DNA or protein synthesis.
[0179] In particular, toxic non-native protein that can be included in engineered microcompartment proteins of the instant disclosure, are toxic proteins or peptides that are non-native to the cell where they are produced and that have a native cellular target substrate which is a native membrane substrate.
[0180] The wording "membrane" as used herein indicates a biological membrane that separates the interior of a cell from the outside environment and can have different structure and configurations in different type of cells as will be understood by a skilled person. In particular, the wording "membrane" as used herein is intended to encompass: i) a cell plasma membrane (also identified as inner membrane in Gram negative bacteria) typically formed by a phospholipid bilayer with embedded proteins, ii) the outer membrane of Gram-negative bacteria formed by a phospholipid bilayer with embedded proteins different in composition from the inner membrane (e.g. rich in lipopolysaccharide), as well as iii) the cell wall, a structural layer that surrounds some types of cells, situated outside the cell membrane and is mainly composed of peptidoglycan (amino acids and sugars). In particular, cell wall can be made of peptidoglycan (also called murein), which is made from polysaccharide chains cross-linked by unusual peptides containing D-amino acids.
[0181] The wording "native membrane substrate" as used herein indicates a compound that is naturally located in the membrane of a cell in the sense of the disclosure and in particular in the membrane of the cell where the toxic non-native protein is expressed or to be expressed. Native membrane substrates comprise proteins, peptidoglycans, and lipids located in the plasma membrane, inner membrane, outer membrane or cell wall of a cell in the sense of the disclosure.
[0182] An exemplary native membrane substrate that can be targeted by toxic proteins herein described are peptidoglycan and lipopolysaccharide (LPS) biosynthesis proteins, which are enzymes such as MraY, LpxK, KdtA, LpxL, LpxM, MraG, FtsW catalyzing biosynthesis of peptidoglycans of the cell wall and LPS in the outer membrane. In particular MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate [13]) Non-native toxic proteins such as LysE react with peptidoglycan with resulting damage to the cell wall and to the cell. LpxK is a gene encoding tetraacyldisaccharide 4'-kinase, an enzyme that phosphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipopolysaccharide lipid A. This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. KdtA is a gene encoding 3-deoxy-D-manno-octulosonic acid transferase, which is involved in lipopolysaccharide (LPS) biosynthesis. This enzymes catalyzes the transfer of two 3-deoxy-D-manno-octulosonate (Kdo) residues from CMP-Kdo to lipid IV(A), the tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A. LpxM is a gene encoding Lipid A biosynthesis myristoyltransferase, an enzyme that catalyzes the transfer of myristate from myristoyl-acyl carrier protein (ACP) to Kdo(2)-(lauroyl)-lipid IV(A) to form Kdo(2)-lipid A. In vitro, the protein can acylate Kdo(2)-lipid IV(A), but the acylation of (Kdo)2-(lauroyl)-lipid IV(A) is about 100 times faster. In vitro, the protein can use lauroyl-ACP but displays a slight kinetic preference for myristoyl-ACP. LpxL is a gene encoding Lipid A biosynthesis lauroyltransferase, an enzyme that catalyzes the transfer of laurate from lauroyl-acyl carrier protein (ACP) to Kdo(2)-lipid IV(A) to form Kdo(2)-(lauroyl)-lipid IV(A). This enzyme has 10-fold selectivity for lauroyl-ACP over myristoyl-ACP. In vitro, this enzyme can also catalyze a slow second acylation reaction leading to the formation of Kdo(2)-(dilauroyl)-lipid IV(A). FtsW is a gene encoding lipid II flippase FtsW protein, a cell division protein that transports lipid-linked peptidoglycan precursors from the inner to the outer leaflet of the cytoplasmic membrane. This protein is required for localization of Ftsl and may also play a role in the stabilization of the FtsZ ring during cell division.
[0183] Additional, native membrane substrates that can be targeted by native toxic proteins herein described are peptidoglycans comprising a pentapeptide motif A(D/N)LXX (SEQ ID NO:8), where X can be any amino acid with the central position in the pentapeptide motif (also designated as position i) being usually a leucine, position i-2 being usually an alanine and the two subsequent positions (i+1 and i+2) configured so that the side chains of positions i-2 and i point into the hydrophobic interior of the protein while the side chains of positions i-1, i+1 and i+2 are exposed on the surface of the proteins. Those peptidoglycans can be targeted for example by non-native lysin proteins with a peptidase domain which can be identified for example using a BLAST search on NCBI. For instance, Ply500 has a pfam02557: VanY: D-alanyl-D-alanine carboxypeptidase motif which would react with a pentapeptide motif in a peptidoglycan. Additional lysins can be identified by a skilled person upon reading of the present disclosure.
[0184] Further native membrane substrates that can be targeted by native toxic proteins are peptidoglycans comprising a sugar motif, such as GlcNAc-X-GlcNAc with X being any amino acid and other sugar motifs identifiable by a skilled person. These native membrane substrates can be targeted by non-native lysins proteins having an amidase domain, which can also be identified for example using a BLAST search, and additional lysins identifiable by a skilled person.
[0185] Additional native membrane substrates that can be targeted by native toxic proteins herein described are phospholipids in the inner membrane. In those embodiments, one or more non-native toxic proteins can bind to lipid and inhibit proper structure of the lipid bilayer membrane, causing holes to form in the membrane. Examples of toxic proteins targeting phospholipids are AMPs having alpha helical or beta-sheet that disrupt inner membrane such as cecropin, magainin, melittin, and protegrin I.
[0186] Further native membrane substrates that can be targeted by native toxic proteins are lipids in the outer membrane (e.g., Lipid II and LPS). Examples of toxic proteins targeting lipids of the outer membrane comprise cationic antimicrobial peptides such as cecropin P1, defensins, and nisins.
[0187] Additional native membrane substrates that can be targeted by native toxic proteins herein described are outer membrane proteins such as integral outer membrane proteins folding into antiparallel beta-barrels. (e.g. proteins belonging to the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA). Examples of toxic proteins targeting lipids of the outer membrane proteins are cationic antimicrobial peptides. An example is inhibition of OmpF porin by HP(2-20) peptide. Additional cationic antimicrobial peptides expected to be found in ([14] [15]).
[0188] In embodiments herein described, the non-native proteins are expressed in constructs where one or more non-native proteins is fused to at least one encapsulin protein herein described to form protein to provide an engineered microcompartment protein, in which the non-native protein can be later released by cleaving from the engineered microcompartment protein. Some non-native proteins have an extended, non-helical structures (e.g., LL-37, Apidaecin Ia) while others have an alpha helical structure (e.g., HBCM2--which is a hybrid of cecropin and melittin, which are both alpha helical).
[0189] In some embodiments, "toxic non-native protein" that can be used as target protein in engineered microcompartment protein and in related cells compositions methods and systems of the instant disclosure comprise antimicrobial peptides targeting cell membrane, proteases targeting proteins in a native cell membrane as defined herein, and lysins as will be understood by a skilled person.
[0190] The term "Antimicrobial peptides" or "AMPs", indicates peptides generally less than 200 amino acids and typically between 12 and 50 amino acids, having two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (generally >50%) of hydrophobic residues and having an antimicrobial activity as would be understood by a skilled person. The secondary structures of AMPs typically follow 4 themes, including i) a-helical, ii) .beta.-stranded due to the presence of 2 or more disulfide bonds, iii) .beta.-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain, and iv) extended as would be understood by a skilled person. The final cellular configuration of AMPs typically contains hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. AMPs can cause cell damage by reacting with membrane components to induce membrane permeabilization or with a range of cytoplasmic targets. In some instances, amino acid composition, amphipathicity, cationic charge and size of AMPs allow them to attach to and insert into membrane bilayers to form pores by `barrel-stave`, `carpet` or `toroidal-pore` mechanisms. In some instances, AMPs can bind target intracellular molecules which are crucial to cell viability thus resulting in cell damage through inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of DNA, RNA, and protein synthesis, and/or inhibition of enzymes identifiable by a skilled person. In general, the antimicrobial activity of these peptides is determined by measuring the minimal inhibitory concentration (MIC), which is the lowest concentration of drug that inhibits bacterial growth. Antimicrobial peptides have been demonstrated to kill Gram negative and Gram-positive bacteria, enveloped viruses, fungi and even transformed or cancerous cells. In some embodiments, the AMPs herein used for the constructing engineered microcompartment proteins have an extended or alpha helical structure.
[0191] In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise cationic AMPs that target phospholipids in the inner membrane, such as cecropin, magainin, melittin, and protegrin I, or derivatives thereof.
[0192] In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise cationic AMPs that target native outer membrane proteins, such as HP(2-20) peptide capable of targeting and inhibiting OmpF porin as well as SMAP-29 and CAP-18 both capable of targeting and inhibiting outer membrane protein I (Oprl).
[0193] In particular, the term "cecropins" indicate AMPs of about 31-37 amino acid residues having alpha helical conformation and being capable of targeting native membrane substrates of both Gram-positive and Gram-negative bacteria. Cecropins isolated from insects other than Hyalophora cecropia (Cecropia moth) are also known as bactericidin, lepidopterin, sarcotoxin, and additional names identifiable by a skilled person. Exemplary cecropin comprise Cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK SEQ ID NO: 20) having a secondary structure with two a helices, and being capable of forming a ion channels at low peptide to lipid ratios and pores at high peptide to lipid ratios as will be understood by a skilled person. Exemplary cecropins also comprise: Cecropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL SEQ ID NO: 21) having two a helices in the secondary structure, CECD from Aedes aegypti (Yellowfever mosquito), Papiliocin (A lepidopteran) from Papilio xuthus an Asian swallowtail butterfly, and Cecropin P1, an antibacterial peptide from Ascaris suum, a parasitic nematode that resides in the pig intestine. Cecropin derivatives comprise peptides modified cecropins (e.g. cecropin A, and cecropin B). In some embodiments, derivatives of cecropins have anticancer properties and are called anticancer peptides (ACPs) ([16] In particular hybrid ACPs based on Cecropin A have been studied for anticancer properties ([17])
[0194] The term "magainins" indicate a class of antimicrobial peptides found in the African clawed frog Xenopus laevis identifiable by a skilled person
[0195] The term "melittin" indicates the principal active component of apitoxin (bee venom), a powerful stimulator of phospholipase A2 as will be understood by a skilled person. Melittin is a peptide consisting of 26 amino acids with the sequence GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:22).
[0196] The term "protegrins" indicates small peptides containing 16-18 amino acid residues. The amino acid composition of protegrins contains six positively charged arginine residues and four cysteine residues. Their secondary structure is classified as cysteine-rich .beta.-sheet antimicrobial peptides, AMPS that display limited sequence similarity to certain defensins and tachyplesins. In solution, the peptides fold to form an anti-parallel .beta.-strand with the structure stabilized by two cysteine bridges formed among the four cysteine residues. Protegrins bind to lipopolysaccharide, a property that may help them to insert into the membranes of gram-negative bacteria and permeabilize them. The term "defensins" as used herein identifies small cysteine-rich cationic proteins found in vertebrates, invertebrates and plants. Defensins have 18-45 amino acids including six to eight conserved cysteine residues. Most defensins function by binding to the microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients.
[0197] The term "nisins" as used herein identifies a polycyclic peptide produced by the bacterium Lactococcus lactis having 34 amino acid residues, including the uncommon amino acids lanthionine (Lan), methyllanthionine (MeLan), didehydroalanine (Dha), and didehydroaminobutyric acid (Dhb). These unusual amino acids are provided by posttranslational modification of the precursor peptide. In these reactions, a ribosomally synthesized 57-mer is converted to the final peptide. The unusual amino acids originate from serine and threonine, and the enzyme-catalyzed addition of cysteine residues to the didehydro amino acids result in the multiple (five) thioether bridges.
[0198] In some embodiments, AMPs that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise HBCM2, HBCM3 and Apidaecin Ia.
[0199] The term "HBCM2" and "HBCM3" refers to hybrid (HB) of silk moth cercropin (C) and bee melittin (M) antimicrobial peptides. These are residue optimized peptides that have high efficacy against Pseudomonas aeruginosa [18, 19]. They originate from cercropin from silk moth and melittin from bees, which are both alpha helical in secondary structure and therefore, HBCM2 and HBCM3 are also thought to be alpha helical in structure.
[0200] The term "Apidaecin Ia" (AP) refers to a series of small, proline-rich, 18- to 20-residue peptides produced by insects. They are the largest group of Pro-rich antimicrobial peptides known to date. Structurally, apidaecins consist of two regions, the conserved region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many Gram-negative bacteria by special antibacterial mechanisms. The mechanism of action by which apidaecins kill bacteria involves an initial non-specific binding of the peptides to an outer membrane (OM) component. This binding is followed by invasion of the periplasmic space, and by a specific and essentially irreversible combination with a receptor/docking molecule that may be a component of a permease-type transporter system on inner membrane (IM). In the final step, the peptide is translocated into the interior of the cell where it meets its ultimate target. Evidence that apidaecins are non-toxic for human and animal cells is a prerequisite for using them as novel antibiotic drugs.
[0201] The term "protease" (also called a peptidase or proteinase or proteolytic enzyme) indicates any enzyme that performs proteolysis, (begins protein catabolism) by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. Proteases can be classified into seven broad groups based on the amino acid at the (protease's) active site used to perform a nucleophilic attack on the substrate: Serine proteases--using a serine alcohol; Cysteine proteases--using a cysteine thiol; Threonine proteases--using a threonine secondary alcohol; Aspartic proteases--using an aspartate carboxylic acid; Glutamic proteases--using a glutamate carboxylic acid; Metalloproteases--using a metal, usually zinc; Asparagine peptide lyases--using an asparagine to perform an elimination reaction (not requiring water), as would be understood by a skilled person. In particular, Aspartic, glutamic and metallo- proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it. Serine, threonine and cysteine proteases use a nucleophilic residue in attack (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme. Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, and elastase). Some proteases can be promiscuous and react with wide range of protein substrates. This is the case for example of digestive enzymes such as trypsin which have to be able to cleave the array of proteins ingested into smaller peptide fragments. Promiscuous proteases typically bind to a single amino acid on the substrate and so only have specificity for that residue. For example, trypsin is specific for the sequences . . . K\ . . . or . . . R\ . . . (`\`=cleavage site). Some proteases are specific and only cleave substrates with a certain sequence or amino acid structure. Proteases, being themselves proteins, can be cleaved by other protease molecules, sometimes of the same variety. This acts as a method of regulation of protease activity. Some proteases are less active after autolysis (e.g. TEV protease) whilst others are more active (e.g. trypsinogen). Specific proteases targeting native membrane substrates are expected to be usable in methods and systems described herein.
[0202] In some embodiments herein described, the proteases inserted as target protein in engineered microcompartment proteins herein described are generally non-specific in their recognition site, meaning their recognition sequence are recognized by a large number of protein substrates. A lack specificity of a protease can be due to a short recognition sequence and/or promiscuity of the protease. Proteases can also be used for cleaving toxic non-native proteins from the engineered microcompartment proteins. Such proteases used for cleaving toxic non-native proteins would be highly specific for a unique recognition sequence that is not commonly found in protein substrates. For instance, TEV protease has a relatively long recognition sequence (ENLYFQ\S(orG)) (SEQ ID NO: 70) that is not commonly found in other proteins. Therefore, it can be used to specifically digest the engineered encapsulin microcompartment and/or release the toxic protein from the encapsulin microcompartment to obtain the toxic protein of interest without resulting in non-specific side products and damage to host cells which in some instance can cause death to the cell. In the case of cellular expression, these specific proteases can digest the encapsulin microcompartments with limited digestion of other cellular proteins that may result in cellular toxicity.
[0203] In particular, specific proteases that target membrane substrates can be compartmentalized with methods herein described. Exemplary specific proteases comprise intramembrane proteases that cleave the transmembrane domain of proteins, such as YaeL from E. coli and SpoIVFB from Bacillus subtilis, additional proteases described in ([20]). In particular, intramembrane proteases such as YaeL (also called RseP) in Escherichia coli play a role in coordinating cell growth and cell division through intramembrane proteolysis of RseA. SpoIVFB is an intramembrane metalloprotease, in Bacillus subtilis that cleaves factors required for sporulation (processing of pro-sigma-K to active SigK). Additional proteases such as endopeptidases that target peptidoglycan. The term "endopeptidases" identifies proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. The relevant peptidase domain can be found by BLAST search on NCBI as will be understood by a skilled person. Additional proteases that target membrane substrates can be identified by a skilled person upon reading of this disclosure.
[0204] The term "lysins", also known as endolysins or murein hydrolases, indicates hydrolytic enzymes produced by bacteriophages in order to cleave the host's cell wall during the final stage of the lytic cycle or natively by bacteria themselves in order to remodel their own cell wall. Usually lysins are monomeric proteins with a 25 to 40 kDa range in size. A notable exception is the streptococcal PlyC endolysin, which is 114 kDa and composed of two different gene products, PlyCA and PlyCB, with a ratio of eight PlyCB subunits for each PlyCA in its active conformation as will be understood by a skilled person. Lysins comprise an at least one domain catalyzing the hydrolysis of peptidoglycan and a domain binding to the cell wall substrate. In lysins, the catalytic domain is responsible for the cleavage of peptidoglycan bonds, and can be one of the following five types of lysin catalytic domain: Endo-P-N-acetylglucosaminidase, N-acetylmuramidase (lysozyme-like), Endopeptidase, N-acetylmuramoyl-L-alanine amidase, .gamma.-D-glutaminyl-L-lysine endopeptidase identifiable by a skilled person. In lysins, the cell-binding domain (CBD) binds to a specific substrate found in the host bacterium's cell wall, usually a carbohydrate. In contrast to the catalytic domain, the cell-binding domain is variable, which allows a great specificity and decreases bacterial resistance. Binding affinity to the cell wall substrate tends to be high, possibly so as to sequester onto cell wall fragments any free enzyme, which could compete with phage progeny from infecting adjacent host bacteria. In lysins usually, two or more different catalytic domains are linked to a single cell-binding domain. This is typical in many staphylococcal lysins as well as the streptococcal PlyC holoenzyme, which contains two catalytic domains. Catalytic domains are highly conserved in phage lysins of the same class. In monomeric lysins, the catalytic domain is typically at the N-terminal end of the protein and the cell binding domain is located at the C-terminal end of the protein and the two domains are separated by a short linker region. Target cellular substrate of lysins are peptidoglycans, which consists of cross-linked amino acids and sugars which form alternating amino sugars: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Endo-.beta.-N-acetylglucosaminidase lysins cleave NAGs while N-acetylmuramidase lysins (lysozyme-like lysins) cleave NAMs. Endopeptidase lysins cleave any of the peptide bonds between amino acids, whereas N-acetylmuramoyl-1-alanine amidase lysins (or simply amidase lysins) hydrolyze the amide bond between the sugar and the amino acid moieties. Finally, the recently discovered .gamma.-d-glutaminyl-1-lysine endopeptidase lysins cleave the gamma bond between D-glutamine and L-lysine residues. Lysins typically target one of the five bonds in peptidoglycan (murein), the main component of bacterial cell walls, which allows the release of progeny virions from the lysed cell in the case of phage lysins and the remodeling of cell wall in the case of native bacterial lysins. These enzymes are being used as antibacterial agents due to their high effectiveness and specificity in comparison with antibiotics, which are susceptible to bacterial resistance.
[0205] In some embodiments, lysins that can be produced or provided in a cell according to methods and systems of the disclosure and related cell and compositions comprise lysozyme-like lysins, such as Cpl-1 and Cpl-7 that target S. pneumoniae peptidoglycan, amidase lysins, such as PlyPSA that targets L. monocytogenes peptidoglycan and endopeptidases that target the pentapeptide motif of peptidoglycan, such as Ply500 that targets L. monocytogenes peptidoglycan and additional lysins described in reference ([21]).
[0206] In particular, the term "lysozyme like lysins" indicates lysins with a catalytic N-acetylmuramidase (lysozyme-like) domain, the term "amidase lysins" identifies with an amidase domain such as amidase 3 domain as shown in the website ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=119407 at the date of filing of the instant disclosure.
[0207] In some embodiments herein described, toxic non-native proteins are non-native proteins or peptides that act within the membrane either by direct interaction/disruption of the membrane or through inhibition of membrane biosynthesis proteins. Examples of such toxic non-native proteins include LysE protein from phiX174 bacteriophage and antimicrobial peptides [22]. LysE protein binds to and inhibits the peptidoglycan biosynthesis protein MraY located in the bacterial membrane, thus resulting in cell lysis, and antimicrobial peptides targeting the bacterial cell membrane, and/or targeting other cellular target substrate other than DNA [23].
[0208] In some embodiments herein described, target proteins introduced in engineered microcompartment protein are non-native AMPs lacking disulfide bonds, such as cecropin, melittin, and apidaecin AMPs.
[0209] In some embodiments described, the toxic non-native proteins introduced as target proteins of engineered microcompartment protein are pro-rich antimicrobial peptides. Examples of toxic non-native proteins that can be used in engineered microcompartment proteins, engineered microcompartment, related vectors, cells compositions methods and systems of the disclosure include apidaecin and antimicrobial peptides which target membrane substrates, and are harmful to bacterial cells (see Example 1).
[0210] In embodiments according to the instant disclosure a microcompartment protein is engineered to introduce at least one non-native protein having a sequence up to 80 amino acids in length at the N-terminus of the first segment of the P-domain of the microcompartment protein, together with at least one protease cleavage site inserted between the C-terminus of the non-native protein and the N-terminus of the first segment of the P-Domain of the microcompartment protein.
[0211] FIG. 8 shows another exemplary engineered microcompartment protein (SEQ ID NO: 23) comprising Apidaecin Ia peptide fused to the N-terminus of an encapsulin protein from M xanthus through a TEV protease cleavage site and a linker region.
[0212] In some embodiments, the target protein can be fused to the N-terminus of the encapsulin protein or encapsulin-like proteins. For example, the target protein can be inserted in an insertion region between the first and second position (the M in X1 and E in X2) of the encapsulin shell protein having the SEQ ID NO: 47 (see Example 12-15). In some of those embodiments, a linker can be comprised within the insertion region placed between the non-native protein and the N-terminus of the encapsulin protein. In some embodiments, the linker comprises a protease cleavage site in order to enable later cleavage of the target protein from encapsulin via a protease that specifically targets at the protease cleavage site. Exemplary recognition sequences and cleavage sites of proteases include the ones shown in Table 2 as well as others identifiable by a person skilled in the art.
[0213] Exemplary engineered microcompartments designed to include a peptide fused to the N-terminus of an engineered encapsulin protein through a protease cleavage site and a linker region are described in Example 17 of the present disclosure.
[0214] In some embodiments, the engineered protein is configured so that cleavage of the target protein from encapsulin via a protease that specifically targets at the protease cleavage site results in a target protein having N-terminal residues of the cleavage site attached to its C-terminus. In some of those embodiments the N-terminal residues of the cleavage site can be undesired as for example they can interfere with the activity of the cleaved target protein.
[0215] In those embodiments, the engineered microcompartment protein can be configured to include a proline residue between the N-terminus of the protease cleavage site and the C-terminus of the target protein. In those embodiments the N-terminal residues of the cleavage site attached to the target protein can be digested with carboxypeptidase that proteolyzes from the C-terminus of the target protein but does not have peptidase activity at proline residues (see Example 27).
[0216] Exemplary embodiments wherein insertion of a proline residue between the N-terminus of the protease cleavage site and the C-terminus of the target protein can be desired are provided by constructs including protease cleavage sites with the overall charge of their N-terminal residues interferes with the proper folding and/or activity of the target protein. Examples of these protease cleavage sites include: the enterokinase protease cleavage site (DDDDK SEQ ID NO: 71), the TEV protease cleavage site (ENLYFQ/G SEQ ID NO: 72), and the HRV-3C protease cleavage site (LEVLFQ/GP SEQ ID NO: 73) whose N-terminal residues have an overall negative charge, as well as the thrombin protease cleavage site (LVPR/GS SEQ ID NO: 74) whose N-terminal residues have an overall positive charge. These N-terminal residues may interfere with a target peptide depending on the configuration of the target peptide.
[0217] In those embodiments, a proline can be inserted between the N-terminal residue of the protease cleavage site (e.g., N-terminal D residue of the enterokinase protease cleavage site) and the C terminus of the target protein or a protease cleavage site with an overall net neutral charge can be selected in order to retain or improve the activity of the target peptide in its native state. Examples of such protease cleavage site include the recognition sequence for the Factor Xa IEGR (Table 2)
[0218] In general in embodiments, wherein a target peptide has a configuration which is known or expected to be incompatible with one or more protease cleavage sites (e.g. because of negatively or positively charged N terminal residues of the protease cleavage site or other incompatibilities), replacement of the protease cleavage site with an alternative protease cleavage which does not interfere with the target protein of interest can be performed. In the alternative placement of a proline in the construct between the N-terminus of the cleavage site and the C terminus of the target protein, can also be performed to allow digestion of the N-terminal residues of the cleavage site attached to the C-terminus of the target protein.
[0219] Protease cleavage sites can be tested to determine if following cleavage, the residual protease cleavage site on the C-terminus of the target protein interferes with the target protein activity by comparing the activity of the target protein with and without the residual protease cleavage site at its C-terminus. The target protein with and without the residual protease cleavage site at its C-terminus can be obtained by chemical synthesis methods (e.g., solid phase peptide synthesis) via commercial sources (e.g., Elim biopharmaceuticals). Activity of the target protein can be determined by an appropriate enzymatic or cell inhibition assay.
[0220] As an alternative, the target protein fused to a proline residue followed by the protease cleavage site followed by the engineered microcompartment protein can be translated in the cytoplasm of a cell and purified using methods identifiable by a skilled person. The purified material can be digested with the appropriate protease to obtain the target protein with the residual protease cleavage site on its C-terminus. The target protein with the residual protease cleavage site can be further digested with carboxypeptidase to obtain the target protein with a residual proline at its C-terminus. The activities of the target protein with the residual protease cleavage site versus the residual proline can be compared to each other as well as a chemically synthesized target protein with no residual amino acids at its C-terminus. This method will determine if any residual amino acids at the C-terminus of a target protein affects its activity.
[0221] In some embodiments, one or more protease sites can also be inserted within the encapsulin shell protein to enable full digestion of the encapsulin cage and thus full release of the non-native protein. The protease cleavage sites can be inserted within 1-8 amino acids adjacent to the C-terminus of the E-loop of the SEQ ID NO:1 at the .beta.3 .beta.-sheet region close to the P-domain. In some other embodiments, the protease cleavage sites can be inserted within 1-8 amino acids adjacent to the N-terminus of the A-domain at the surface-exposed region (see Examples 12-17).
[0222] In some embodiments, the engineered microcompartment protein can further include one or more tags inserted in the engineered microcompartment protein.
[0223] The term "tag" as used herein means protein tags comprising peptide sequences introduced onto a recombinant protein. Tags can be removable by chemical agents or by enzymatic means, such as proteolysis or splicing. Tags can be attached to proteins for various purposes: Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. These include chitin binding protein (CBP), and the poly(His) tag. The poly(His) tag is a widely-used protein tag; it binds to metal matrices. Chromatography tags can be used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag. Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Protein tags can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging). Tags can be combined, in order to connect proteins to multiple other components. However, with the addition of each tag comes the risk that the native function of the protein may be abolished or compromised by interactions with the tag. Therefore, after purification, tags are sometimes removed by specific proteolysis (e.g. by TEV protease, Thrombin, Factor Xa or Enteropeptidase).
[0224] Exemplary tags comprise the following, among others known to persons skilled in the art: Peptide tags, such as: AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE (SEQ ID NO:24)); Calmodulin-tag, a peptide that can be bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO:25)); polyglutamate tag, a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE (SEQ ID NO:26)); E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR (SEQ ID NO:27)); FLAG-tag, a peptide recognized by an antibody (DYKDDDDK (SEQ ID NO:28)); HA-tag, a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA (SEQ ID NO: 29)); His-tag, typically 5-10 histidines that can be bound by a nickel or cobalt chelate (HEIHEIHH (SEQ ID NO:30)); Myc-tag, a peptide derived from c-myc recognized by an antibody (EQKLISEEDL (SEQ ID NO:31)); NE-tag, a novel 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES (SEQ ID NO:32)) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, a peptide derived from Ribonuclease A (KETAAAKFERQHMDS (SEQ ID NO:33)); SBP-tag, a peptide which binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:34)); Softag 1, for mammalian expression (SLAELLNAGLGGS (SEQ ID NO:35)); Softag 3, for prokaryotic expression (TQDPSRVG (SEQ ID NO:36)); Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK (SEQ ID NO:37)); TC tag, a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC (SEQ ID NO:38)); V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST (SEQ ID NO:39)); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK (SEQ ID NO:40)); Xpress tag (DLYDDDDK (SEQ ID NO:41)); Covalent peptide tags such as: Isopeptag, a peptide which binds covalently to pilin-C protein (TDKDMTITFTNKKDAE (SEQ ID NO:42 SpyTag, a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK (SEQ ID NO:43)); SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK (SEQ ID NO:44)).
[0225] In embodiments described herein, any of the tags of SEQ ID NO:24-44, and other tags known to those skilled in the art, can comprise one or more amino acid substitutions, insertions, or deletions that do not alter the function of the tag, and can further comprise one or more additional amino acids, up to a maximum tag length of 100 amino acids. In preferred embodiments, the tag comprises up to a maximum of 20 amino acids in length.
[0226] In some embodiments, the protein tag can be a polyhistidine tag. A polyhistidine-tag is an amino acid motif in proteins that typically consists of six histidine (His) residues typically, often at the N- or C-terminus of the protein. It is also known as hexahistidine-tag, 6.times. His-tag, His6 tag and by the trademarked name His-tag (registered by EMD Biosciences). The total number of histidine residues can vary in the tag. N- or C-terminal his-tags can also be followed or preceded, respectively, by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endopeptidases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. Fluorescent hexahistidine CyDye tags are also available. These use Nickel covalent coordination to EDTA groups attached to fluorophores in order to create dyes that attach to the polyhistidine tag. This technique has been shown to be effective for following protein migration and trafficking. This technique can also be effective in order to measure distance via Fluorescent Resonance Energy Transfer.
[0227] In some embodiments, engineered microcompartment proteins comprise tags up to 8 amino acids in length inserted within the engineered microcompartment proteins as described herein. Exemplary tags include peptide tags such as AviTag, E-tag, FLAG-tag, His6-tag, Strep-tag and as well as other known to persons skilled in the art.
[0228] [00193]M some embodiments, the tags can be inserted within the A-domain of the encapsulin protein. In particular, the tags can be inserted within 1-8 residues adjacent to the N-terminus of the A-domain of SEQ ID NO: 47 (see Example 12 and 18). In some embodiments, the tags can be inserted in the E-loop.
[0229] In some embodiments herein described, an insertion region comprising at least one first protease cleavage site and the insertion region comprising the at least one second protease cleavage site have independently lengths up to 22 amino acids including any linker, protease cleavage sites and tags.
[0230] The term "linker" as used herein indicates a short peptide sequences that occur between protein domains. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. In particular, in engineered microcompartment protein of the disclosure linkers are typically peptide of 2 to 5 residues in combination with a protease cleavage site, a target protein, and/or a tag as will be understood by a skilled person upon reading of the present disclosure. The linker between the protease cleavage site and the encapsulin protein can comprise at least one glycine residue. The linker can be as short as two amino acids in length.
[0231] Exemplary linkers include GGGGS (SEQ ID NO:75), GSGSG (SEQ ID NO:76), GGGG (SEQ ID NO:77), GGG(SEQ ID NO:78), GG(SEQ ID NO:79), GS(SEQ ID NO:80), GSGS(SEQ ID NO:81), GGGS(SEQ ID NO:82), GGS(SEQ ID NO:83), GTS (SEQ ID NO:84), GGGGT (G.sub.4T) (SEQ ID NO: 85) and others identifiable by a person skilled in the art.
[0232] In some embodiments wherein a target protein is packaged within a cage-forming encapsulin construct, the presence of a linker between the protease cleavage site and the N-terminus of the engineered encapsulin is preferred. In some embodiments insertion of a linker of at least 5 residues in length containing at least 1 glycine residue between the protease cleavage site and the encapsulin protein can facilitate the release of the target protein following the protease digestion (Example 19).
[0233] In some embodiments, the engineered microcompartment proteins can be designed to be cage forming engineered microcompartment proteins or non cage forming engineered microcompartment proteins as described herein.
[0234] Preferred cage forming engineered microcompartment proteins typically comprise one protease cleavage site between the C-terminus of the peptide and N-terminus of the engineered encapsulin. Preferred cage forming engineered microcompartment proteins typically also comprise a linker between the first protease cleavage site and the N-terminus of the engineered encapsulin wherein the linker is preferably flexible (containing at least 1 glycine residue) and greater than 2 amino acids in length. A typical linker is a penta-glycine linker (GGGGG SEQ ID NO: 143). The linker length is selected not to exceed a length that would cause the (peptide)+(protease cleavage site)+(linker) to be >80 amino acids. Preferred cage forming engineered microcompartment proteins further comprise one protease cleavage site within the loop region of the E-loop domain. This region is the unstructured region between beta-strands .beta.2 and .beta.3. Examples are insertions following residues V57 and D60 in T. maritima encapsulin. An additional protease cleavage site can also be placed in the A-domain within the beta-strand .beta.5 and the subsequent unstructured region. An example is insertion following residue K138 in T. maritima encapsulin. The addition of this site may improve the kinetics of peptide release.
[0235] Preferred cage forming microcompartment proteins can be advantageously used for example when production of a protease-sensitive peptide where the cage protects the peptide from proteolysis during expression; storage of a peptide to protect it from proteolysis within cells or in vitro as purified protein are desired.
[0236] Preferred cage forming microcompartment proteins are also expected to be advantageously used when improved secondary structure formation (e.g., disulfide bond formation) during peptide expression is desired.
[0237] Preferred non-cage forming engineered microcompartment proteins typically comprise one protease cleavage site between the C-terminus of the peptide and N-terminus of the engineered encapsulin. Preferred non-cage forming engineered microcompartment proteins typically also comprise a linker between the first protease cleavage site and the N-terminus of the engineered encapsulin. The linker is preferably flexible (containing at least 1 glycine residue) and can be as short as 2 amino acids in length. The linker is selected not to exceed a length that would cause the (peptide)+(protease cleavage site)+(linker) to be >80 amino acids. Preferred non-cage forming engineered microcompartment proteins typically further comprise one protease cleavage site in beta-strand .beta.3 of the E-loop domain, within 9 amino acids of the N-terminus of the adjacent P-domain fragment. An example is an insertion following residue K71 in T. maritima encapsulin. An additional protease cleavage site followed by an affinity purification tag can also be placed in the A-domain within the beta-strand .beta.5 and the subsequent unstructured region. An example is insertion following residue K138 in T. maritima encapsulin. Here, the affinity tag can be used for purification of the construct, which is accessible in a non-cage forming encapsulin mutant.
[0238] Preferred non-cage forming microcompartment proteins can be advantageously used for example when_high yield production and release of a peptide--purification shall be done rapidly and in the presence of protease inhibitors to prevent any proteolysis. Non-cage forming microcompartment proteins are also expected to be advantageous when_an improved secondary structure formation (e.g., disulfide bond formation) during peptide synthesis is desired.
[0239] A skilled person will be able to identify how to configure an engineered microcompartment protein of the disclosure based on the target protein and other features of the related production. For example, in some embodiments wherein the target protein is protease-sensitive, the insertion of the one or more protease sites within the encapsulin shell protein is preferably selected to provide a cage forming engineered microcompartment protein to maximize incorporation of expressed peptide into an encapsulin cage such that the cage-forming encapsulin construct can protect the target protein from proteolysis during expression or for the storage of the expressed target protein within cells or in vitro as purified protein. The cage-forming encapsulin construct is also expected to facilitate improved secondary structure formation, such as disulfide bond formation, during peptide expression.
[0240] In some of these embodiments, the protease cleavage site can be preferably provided within the unstructured loop region of the E-loop domain. In some exemplary embodiments exemplified in Example 18 the protease cleavage site can be inserted following residues V57 and D60 in T. maritima encapsulin. Insertion of a protease site following K138 in T. maritima (corresponding to the region within 2-24 amino acids of the N-terminus of the A-domain (the A-domain within the beta-strand .beta.5 and the subsequent unstructured region) also maintains cage formation.
[0241] In embodiments, wherein the target protein is protease-sensitive, protease cleavage sites can be selected to provide a non-cage forming engineered microcompartment protein in embodiments wherein purification of the non-cage forming engineered microcompartment protein is performed under conditions to prevent proteolysis, such as in the presence of additives that prevent proteolysis such as B-PER II detergent or protease inhibitors. In addition, or in the alternative purification can be performed within the timeframe when degradation of less 50% of the protein is detected, typically within 16 hrs. or less. Methods to detect protein degradation comprise densitometry performed on Western blot or SDS-PAGE of the proteins in the lysate comprising the protein and additional techniques identifiable by a skilled person. In some of those embodiments, a protease cleavage site can be placed at the C terminus of E-Loop domain or within 1-8 amino acids adjacent to the C-terminus of the E-loop domain within beta-strand .beta.3. An exemplary embodiment is an insertion following residue K71 in T. maritima encapsulin (see Example 18).
[0242] In some embodiments wherein fast release of the target protein is desired, the insertion of the one or more protease sites within the encapsulin shell protein is preferably selected to provide a non-cage forming engineered microcompartment protein comprising at least one protease cleavage site in the beta-strand .beta.3 of the E-loop domain or in combination with at least one protease cleavage site in the unstructured region of the E-Loop and/or the A domain within the beta-strand .beta.5 and the subsequent unstructured region. The second insertion can improve the kinetics of peptide release in some embodiments. An exemplary embodiment is provided by constructs comprising a protease cleavage site following residue K71 in T. maritima encapsulin (see Example 18).
[0243] In some of these embodiments wherein a high yield production and release of a target peptide is desired, a protease cleavage site can be selected within 0-8 amino acids residues adjacent to the C-terminus of the E-loop of the encapsulin protein within the beta-strand .beta.3. In an exemplary embodiment the protease cleavage site is inserted following residue K71 in T. maritima encapsulin (Example 18). Such insertion site can effectively disrupt cage formation (see Example 19).
[0244] In several embodiments, the non-native protein to be produced or provided with methods of the disclosure comprise proteins or peptides that can be used as chemotherapeutic drugs in treating cancer to kill, inhibit growth or halt the replication and/or spread of cancerous cells in a patient. In some of those embodiments, the non-native protein or peptides are AMPS that can be used in cancer treatment.
[0245] In methods and systems herein described and related cell and compositions, one or more target proteins and in particular one or more proteins non-native to a bacterial cell are expressed in said cell within at least one engineered microcompartment protein to form at least one engineered microcompartment comprising the one or more toxic non-native proteins within the microcompartment.
[0246] The term "express" as used herein with reference to proteins or peptide indicates the way in which proteins or peptides are synthesized, modified and regulated in living organisms. Typically, protein expression includes DNA transcription, RNA processing, translation, and post-translational modification of a protein as will be understood by a skilled person. In particular, the term protein expression refers the process of generating a specific protein within a cell and includes the transcription of the recombinant DNA to messenger RNA (mRNA) and the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations
[0247] Expression system for protein production comprise a combination of an expression vector, its cloned DNA, and the host cell for the vector that provide a context to allow a non-native gene function in a host cell, that is, produce proteins. Example expression systems are 1) BL21(DE3) host cells that express protein from an expression vector that contains a pT7 phage promoter; and 2) BL21 host cells that express protein from expression vectors that contain pT5 or pRha promoters. Additional expression systems and related host cells, vector and promoters are identifiable by a skilled person
[0248] The term "cell" or " bacterial cell" as used herein indicates a bacterial cell with bacteria indicating several prokaryotic microbial species which include but are not limited to Gram-positive bacteria, Proteobacteria, Cyanobacteria, Spirochetes and related species, Planctomyces, Bacteroides, Flavobacteria, Chlamydia, Green sulfur bacteria, Green non-sulfur bacteria including anaerobic phototrophs, Radioresistant micrococci and related species, Thermotoga and Thermosipho thermophiles. More specifically, the wording "Gram positive bacteria" refers to cocci, nonsporulating rods and sporulating rods, such as, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus and Streptomyces. The term "Proteobacteria" refers to purple photosynthetic and non-photosynthetic gram-negative bacteria, including cocci, nonenteric rods and enteric rods, such as, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema and Fusobacterium. Cyanobacteria, e.g., oxygenic phototrophs.
[0249] In embodiments herein described cytotoxic proteins are expressed inside engineered microcompartment proteins, the engineered microcompartment proteins can shield the cells from toxicity of the cytotoxic proteins, allowing the cells to grow and thus produce more cytotoxic proteins.
[0250] In some embodiments, methods of the present disclosure comprises introducing into the cell at least one polynucleotide encoding at least one engineered microcompartment protein herein described, the at least one polynucleotide operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the cell, the at least one engineered microcompartment protein capable of assembling with one or more same and/or different microcompartment proteins to form at least one empty microcompartment within the cell.
[0251] As used throughout, "regulatory elements" are regions of non-coding DNA which regulate the transcription of nearby genes. Examples of regulatory elements are promoters and enhancers. Enhancers are regions of DNA that can be bound with proteins (activators) to activate transcription of a gene or transcription. Promoters are regions of DNA that initiate transcription of a particular gene. In the embodiments described, types of promoters used are over-expression promoters, low-level promoters and tunable promoters. Tunable promoters are not constitutive and can be activated or inactivated as a result of culturing conditions and/or additional elements. In some embodiments, tunable promoters are activated in the presence of a compound introduced into the culture media. Examples of tunable promoters includepRha. In the embodiments described, selection of a promoter is determined by several factors including, but not limited to, the nature of the protein being expressed and the desired expression level of the expressed protein. In the embodiments described, low-level promoters are used when the toxic non-native protein is not efficiently localized to the interior of a microcompartment so as to reduce toxicity to a cell from accumulation of the toxic non-native protein in the cell. In the embodiments described, the use of tunable promoters is used to express a protein at a certain level and/or time during culturing of the cell. In the embodiments described, the type of promoter used is influenced by the interplay between the microcompartment proteins and the toxic non-native proteins.
[0252] Accordingly, selection of the appropriate regulatory elements and the at least one polynucleotide can be performed with procedures identifiable by a skilled person.
[0253] As used throughout, "operably linked" is defined as a functional linkage between two or more elements. In particular, the term "operably linked" or "operably connected" indicates an operating interconnection between two elements finalized to the expression and translation of a sequence. Functional linkages between elements in the sense of the present disclosure are identifiable by a skilled person. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) comprises a functional link that allows for expression of the polynucleotide of interest. Another example of operable linkage is provided by a control sequence ligated to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked elements are contiguous or non-contiguous and comprise polynucleotides in a same or different reading frame. Additionally, "operably linked" refers to proteins that are linked together wherein the linkage does not impact the function of the individual proteins.
[0254] In embodiments herein described, the engineered microcompartment protein comprises encapsulin or encapsulin-like proteins fused with a toxic non-native protein at the N-terminus of the encapsulin and inserted with one or more protease cleavage sites described herein. In some embodiments, one or more protein tags can be added through genetic modification of the engineered microcompartment proteins herein described in accordance with the present disclosure.
[0255] In some embodiments an engineered microcompartment protein can be introduced in a cell to form an engineered microcompartment according to a method to provide an engineered microcompartment protein in a bacterial cell herein described. The method comprises introducing into the cell at least one polynucleotide encoding at least one engineered microcompartment protein herein described. The at least one polynucleotide operatively linked to one or more first regulatory elements leading to the expression of the at least one engineered microcompartment protein in the cell, the at least one microcompartment protein capable of assembling with one or more same and/or different microcompartment proteins to form at least one microcompartment within the cell. The assembled engineered microcompartment proteins encompass an interior target protein.
[0256] As used throughout, "introducing into the cell" with respect to the polynucleotides refers to inserting a polynucleotide encoding a protein or peptide into a cell or population of cells. One of ordinary skill in the art can readily appreciate that a variety of methods can be used to achieve this such as transformation, transfection, viral transduction and/or injection. In the embodiments described, successful introduction of a polynucleotide into a cell can be assessed by selecting for cells that have taken up the polynucleotide. This is done, for example, by incorporating into the polynucleotide an antibiotic resistance marker against an antibiotic that a cell is typically sensitive to. In some embodiments described, ampicillin resistance genes and kanamycin resistance genes are used on polynucleotides to assess positive insertion of a polynucleotide into a bacterial cell sensitive to ampicillin and kanamycin. Following insertion of a polynucleotide carrying an ampicillin resistance gene, for instance, cells are grown in media containing ampicillin to select for cells that have successfully taken up the polynucleotide.
[0257] Introduction of a polynucleotide can be performed for example by chemical transformation or electroporation or other methods identifiable by a skilled person. In some exemplary embodiments performed in E. coli, chemical transformation can be performed by incubating CaCl.sub.2-treated E. coli cells with the plasmid(s) of interest and heat shocking the cells at 42.degree. C. for an appropriate time period (<60 s) to encourage the cells to take up the plasmid(s). Cells are then diluted with rich medium and incubated at 37.degree. C. to allow for heat shock recovery and expression of one or more antibiotic resistance genes. Cells are then plated on solid-agar medium supplemented with the appropriate antibiotic to select for cells that have taken up the polynucleotide. Similarly, in electroporation, E. coli cells are incubated with the polynucleotide of interest and electroporated at an appropriate voltage to increase cells uptake of the polynucleotide as will be understood by a skilled person. Subsequent steps are the same as for chemical transformation.
[0258] In some embodiments, introducing into the cell the at least one polynucleotide can be performed by introducing an expression vector comprising the at least one polynucleotide and the one or more first regulatory elements in a configuration leading to transcription of the engineered microcompartment protein carried on the expression vector.
[0259] As used throughout, an "expression vector" is a plasmid or virus designed for protein expression in cells. The expression vector is used to introduce a specific gene into a target cell and uses the cell's mechanism for protein synthesis to produce the protein encoded by the gene. In the embodiments described, genes delivered include toxic non-native proteins (such as Ap and HBCM-2) and microcompartment proteins (such as encapsulin of SEQ ID NO: 47). In the embodiments described, a plasmid is engineered to contain regulatory sequences that act as enhancer and/or promoter regions and lead to efficient transcription of the gene carried on the expression vector. Such plasmids also contain selection markers, such as antibiotic resistance markers, to select for cells that have successfully taken up the plasmid. The plasmids herein used for the expression of the engineered microcompartment can also comprise linker sequences, tags and protease cleavage sites. Examples of constructs are pMCY124, pMCY125, and pMCY133 (see Example 12-15).
[0260] In order to introduce the engineered microcompartment proteins to a cell, one or more genes encoding the appropriate engineered microcompartment proteins can be cloned and placed under the control of a promoter (constitutive or inducible) in a given plasmid or other vector of interest containing an antibiotic resistance marker. Genes for coding the non-native protein and encapsulin protein can be place in tandem behind a given promoter with appropriate ribosomal re-initiation sites to ensure all proteins are expressed. The plasmid containing one or more engineered microcompartment genes is then transformed into the host organism by either chemical transformation or electroporation. In the case of a constitutive promoter, microcompartment protein is expressed from the plasmid constantly during growth of the host organism. In the case of an inducible promoter, microcompartment protein is expressed from the plasmid by addition of inducer to growth medium (e.g. IPTG, rhamnose).
[0261] In some embodiments, the one or more first regulatory elements comprise an over-expression promoter, a low-level constitutive promoter or a tunable promoter. For example, in some embodiments, the one or more first regulatory elements can comprise a T7, pTet, pRha or pT5 promoter.
[0262] In some embodiments, the engineered microcompartment proteins can be designed and altered to support functional expression of the cytotoxic non-native proteins to be encapsulated inside one or more engineered microcompartment. For instance, the expression system can be engineered to over-express the encapsulin shell proteins and the non-native proteins to improve yields, thus allowing for higher expression of encapsulated non-native proteins.
[0263] In some embodiments, where the engineered microcompartment proteins are to be overexpressed, expression of one or more appropriate engineered microcompartment proteins can be placed under the control of a highly inducible promoter (e.g., T7, T5, rhamnose). The engineered microcompartment proteins can also be over-expressed from a high copy number plasmid containing an appropriate origin (e.g., pUC) in order to ensure multiple copies of the appropriate genes are expressed. The nucleotide sequence of the microcompartment protein genes can be optimized based on host organism codon usage in order to achieve overexpression as well as according to approaches such as the ones described in reference ([24]) and other approaches identifiable by a skilled person.
[0264] In some embodiments herein described, one or more genes for toxic non-native proteins are fused to one or more genes for encapsulin proteins under the control of an inducible promoter in a given plasmid or other vector of interest to form the at least one polynucleotide encoding an engineered microcompartment protein. The plasmid (or other vector) containing the polynucleotide for the engineered microcompartment protein can be transformed into a host cell by chemical transformation or electroporation and selected for using antibiotic resistance markers or other markers identifiable by a skilled person.
[0265] In some embodiments, at least one polynucleotide comprises one or more polynucleotides encoding for two or more microcompartment proteins.
[0266] In some embodiments, the at least one polynucleotide comprises one or more polynucleotides encoding for encapsulins (see Examples 12-16).
[0267] In some embodiments, the at least one polynucleotide comprises one or more polynucleotides encoding for one or more of non-native proteins. The non-native protein insertions comprise up to 80 amino acids in length (see Examples 15-17).
[0268] In some embodiment herein described, the genes encoding for toxic non-native proteins are operably linked to one another and/or to the genes encoding for encapsulin proteins through a linker. The linker between the non-native protein genes and between the non-native protein gene and the encapsulin gene is configured not to impact the expression of the polynucleotide and the proper folding of the formed engineered microcompartment proteins. The linker also comprises a protease cleavage site specific to a protease, thus allowing the release of the toxic non-native proteins from the encapsulin proteins by proteolysis.
[0269] In some embodiments, the at least one polynucleotide encoding the engineered microcompartment also comprises one or more protease cleavage sites inserted within the genes encoding encapsulins to ensure full digestion of the encapsulin cage and thus full release of the non-native proteins (Examples 12-17).
[0270] In some embodiments, two or more polynucleotides can be introduced in combination, simultaneously or sequentially. Whether to introduce two or more polynucleotides in combination, sequentially or simultaneously depends on the nature of the proteins being expressed from the polynucleotides and the desired results. In the embodiments described, polynucleotides are expressed sequentially, for instance, so as to effectively select for positive insertion of the polynucleotides. For instance, a first polynucleotide encoding for a microcompartment protein and containing an ampicillin resistance gene can be introduced into a group of cells that are sensitive to ampicillin and kanamycin. Following insertion of the first polynucleotide, the cells are cultured in the presence of ampicillin to select for those that have taken up the first polynucleotide. Next, these cells are introduced to a second polynucleotide encoding for a toxic non-native protein and containing a kanamycin resistance gene. Following insertion of the first polynucleotide, the cells are cultured in the presence of kanamycin to select for those that have taken up the second polynucleotide. The resulting cells are thus selected for successful incorporation of both polynucleotides. A similar strategy is taken when the second polynucleotide encodes for a protein that is extremely toxic to the cells and/or inefficiently localized to the interior of a microcompartment. In such an example, a first polynucleotide encoding for a microcompartment protein is introduced before the second polynucleotide encoding for the toxic protein so as to prevent the cell from toxicity following expression of the protein from the second polynucleotide.
[0271] In the embodiments described herein, polynucleotides introduced into a cell encode for a single protein or peptide or several proteins or peptides that function together.
[0272] As used throughout, "conditions" for culturing the cells refer to the various elements required to select and/or maintain cells as well as to the various elements required to obtain the desired amount of protein expression from the polynucleotides. Elements required for these purposes include culture media, antibiotics, chemical inducers to promote expression from a promoter (e.g., i sopropyl-B-D-thiogalactopyranoside, rhamnose, arabinose), CO.sub.2 concentrations, temperature, agitation (in rotations per minute, rpm) and additional factors required to ensure that the proteins are expressed; other elements would be readily appreciated by one of ordinary skill in the art. Additionally, elements include factors that are required for the expressed proteins to function as intended.
[0273] In embodiments herein described, the methods further comprises introducing into the cell at least one second polynucleotide encoding one or more proteases, each protease capable of cleaving at a protease cleavage site inserted within the engineered microcompartment protein, thus releasing the non-native protein from the engineered microcompartment protein. The at least one second polynucleotide is operably linked to one or more second regulatory elements leading to the expression of the at least one protease.
[0274] Exemplary proteases include Human Rhinovirus (HRV) 3C Protease, Enterokinase, Factor Xa, Tobacco etch virus protease (TEV protease), Thrombin and others known to a person skilled in the art.
[0275] In some embodiments, introducing into the cell the at least one second polynucleotide encoding for the proteases is performed by introducing an expression vector comprising the at least one polynucleotide of the at least one second polynucleotide and the one or more second regulatory elements in a configuration leading to transcription of the protease carried on the expression vector.
[0276] In some embodiments, the one or more second regulatory elements comprise a promoter, a low-level constitutive promoter or a tunable promoter. In some embodiments, the one or more second regulatory elements comprise an enhancer.
[0277] In some of those embodiments, the one or more second regulatory elements are different from the one or more first regulatory elements operably linked to the polynucleotide encoding the engineered microcompartment protein. In some embodiment, the second regulatory elements in the polynucleotide encoding the proteases comprise a pRha or pT7 promoter while the first regulatory elements in the polynucleotide encoding the engineered microcompartment proteins comprise pTet or pT5.
[0278] In particular, in embodiments herein described, introducing the second polynucleotide is performed in combination with the introducing of the first polynucleotide to obtain the toxic non-native protein within the cell.
[0279] In some embodiments, proteases can be added directly to the lysed cells or purified engineered microcompartment proteins to release the non-native protein from the microcompartment proteins. In some of these embodiments, the method further comprises purifying the at least one engineered microcompartment protein and adding to the purified engineered microcompartment protein at least one protease targeting the protease cleavage sites of the engineered microcompartment protein to release the non-native target protein from the engineered microcompartment protein to obtain the non-native target protein.
[0280] The target protein can be purified by size exclusion chromatography or by a centrifugal filter with an appropriate molecular weight cutoff in order to separate the target protein from the microcompartment protein based on size. Alternatively, ion exchange chromatography or reverse phase (e.g., C18) chromatography can be used when appropriate as will be understood by a skilled person.
[0281] Purification of the at least one engineered microcompartment protein can be performed rapidly (within 16 hrs. e.g. within 4 h or less) and/or in the presence of suitable additives and/or protease inhibitors to prevent any proteolysis of the engineered microcompartment proteins at this stage. In some other embodiments, the at least one protease can be added to lysed cells expressing the engineered microcompartment proteins herein described. In some embodiments, lysis of the cells can be performed rapidly within 4 h or other suitable time in view of the reaction mixture. Rapid purification of the microcompartment or rapid lysis of the cells are particularly preferred for non cage forming engineered microcompartment protein comprising a protease sensitive target protein as will be understood by a skilled person.
[0282] The term "protease-sensitive" as described herein indicates proteins that are targeted for proteolytic degradation by native proteases in the host cell or cell free reaction where the expression of the protein is performed.
[0283] In embodiments in which an engineered microcompartment protein comprises a proline between the N-terminus of the first at least one protease cleavage site and the C terminus of the target protein the method can further comprise, contacting the purified non native target protein with a carboxypeptidase to allow reaction of the carboxypeptidase with the carboxy terminal residues of the purified non-native target protein.
[0284] First, different protease cleavage sites between the C-terminus of the peptide and the N-terminus of the engineered encapsulin should be tested to determine if there is a residual protease cleavage site that does not interfere with peptide activity.
[0285] These additional contacting of the purified target protein can be performed to achieve activation of a non-active peptide to an active peptide or improvement of the activity of an active peptide.
[0286] In some embodiments, the present disclosure provides a method to express significant amounts of non-native, cytotoxic proteins in a host organism for isolation and production purposes. The method can be applied to proteins of interest that are difficult to produce due to their cytotoxicity to the host organism. In an exemplary method, cytotoxic, non-native proteins are engineered to be encapsulated in encapsulin cage in E. coli in order to prevent their cytotoxicity. As proof of this concept, in some examples herein described, the cytotoxic non-native proteins Ap from honey bee were fused to encapsulins to form engineered microcompartment proteins in the cytoplasm of E. coli, thus shielding the toxicity of Aps from the cells as described in Example 12-15 and 24. In some other examples, the cytotoxic non-native HBCM-2 peptides were fused to encapsulins to form engineered microcompartment proteins thus shielding the toxicity of the peptides from the cells (Example 16-17).
[0287] In some embodiments, the engineered microcompartment proteins formed by fusing cytotoxic target proteins, such as non-native HBCM-2 and AP, to encapsulins can achieve a robust expression while remaining less susceptible to proteolysis in comparison with fusion proteins of the same cytotoxic non-native peptides with other common carrier proteins (Example 24). In particular, the engineered microcompartment proteins formed by fusing cytotoxic non-native target proteins to encapsulins also confer limited toxicity to the host cells even at a high over-expression level (Example 23).
[0288] In some embodiments, the methods herein described can produce a large amount of active, cytotoxic non-native proteins in vivo or in vitro (following cleavage from the engineered microcompartment protein) having the same level of activity as the chemically synthesized proteins (see Example 22). In some embodiments, the present disclosure addresses issues associated with production of non-native, cytotoxic proteins in the host organism E. coli, and can be associated with in vitro purification systems (cell free expression, as described in the www webpage lifetechnologies.com) excretion tags excreting the cytotoxic proteins from E. coli (as described in the www webpage dna20.com), which are used to produce cytotoxic proteins.
[0289] In some embodiments, methods and systems of the disclosure herein described can be used express cytotoxic proteins directly in bacterial microcompartments to minimize toxicity, and/or reduce problems associated with proper folding and secretion associated with secretion tags. The methods, systems and related compositions and cells of the disclosure can result in several embodiments with reduced costs and higher production levels compared to those of prior methods.
[0290] In some embodiments, engineered microcompartment proteins, related engineered microcompartment and related vectors, cells, compositions, methods and systems can be used to introduce in a same engineered microcompartment a same or different target protein.
[0291] An exemplary illustration of steps of methods to provide one or more target proteins such as toxic or non-toxic non-native proteins herein described and in particular Ap are illustrated with reference to exemplary engineered microcompartment proteins expressed in E. coli cells through use of specific regulatory sequences as will be understood by a skilled person.
[0292] A first fusion gene encoding an engineered microcompartment protein is created, the fusion gene comprising a gene of the desired non-native protein fused to the N-terminus of a gene encoding an encapsulin protein. Protease cleavage site such as TEV or thrombin can also be placed between the non-native protein and the encapsulin protein to later cleave off the non-native protein. Protease cleavage sites are also placed within the encapsulin proteins to enable digestion of the encapsulin and thus release of the non-native protein.
[0293] The fusion gene is then placed under the control of an inducible promoter in a plasmid of interest, which is then transformed into an E. coli expression strain (e.g., C43 cells). The transformed E. coli cells then grow in an appropriate medium (LB medium) at an appropriate temperature.
[0294] A second gene encoding a protease can be created and co-expressed with the fusion gene encoding an engineered microcompartment protein.
[0295] The cells are induced for engineered microcompartment protein formation using the inducer for the plasmid containing the microcompartment proteins (e.g., IPTG for the pT5 promoter).
[0296] Alternatively, instead of co-expression of the fusion gene and the protease-encoding gene, proteases can be added directly to lysed cells expressing the engineered microcompartment protein or to purified engineered microcompartment protein.
[0297] In order to isolate the toxic protein of interest inside microcompartments, the cells are harvested after desired induction time and resuspended in an appropriate buffer. The cells are then lysed by an appropriate method including sonication, French press lysis, detergent lysis with lysozyme and other methods identifiable to a person skilled in the art. The cell debris can be removed by centrifugation at 12,000 g, 4.degree. C. for 10 min and supernatant can be collected.
[0298] To collect the toxic protein of interest, appropriate detergent is added to the soluble fraction if necessary (e.g., Empigen BB) and incubated for sufficient time to solubilize desired protein. The soluble fraction is then loaded on an affinity chromatography column. The column is washed and the desired protein is eluted with the appropriate buffers. The fractions containing the desired protein can then be collected and stored as necessary.
[0299] Engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be applied broadly to other cytotoxic proteins for expression and purification from bacterial cells by replacing the gene for Ap with the gene for a cytotoxic protein (toxic, non-native protein) of interest and by replacing the medium and regulatory sequences of E. coli with the ones of a desired bacteria as will be understood by a skilled person. Thus, the methods, systems, cell and compositions of the disclosure have wide applications in the biosciences, where this novel technology could be used for the efficient production of proteins that are normally difficult to produce.
[0300] In embodiments herein described, engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be used to produce a toxic non-native protein, by expressing the toxic non-native protein within BMC in a bacterial cell and by isolating the toxic non-native protein from the bacterial cell.
[0301] In some embodiments, engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure can be used to shield the toxicity of pathway intermediates and increase reaction efficiencies in nature. In those embodiments, a toxic non-native protein can be expressed within a BMC and additional molecule forming the pathway can be provided within the cell. In those embodiments, small molecule substrates and products of the enzymes can passively diffuse in and out of the BMCs via pores in the shell proteins, while pathway intermediates remain sequestered inside the BMCs. In such case, not only the toxic pathway intermediates can be shielded from the host organism, but the local concentration of enzymes and substrates also increases, leading to improved reaction efficiency.
[0302] In several embodiments, the present disclosure provides engineered microcompartment proteins, engineered microcompartments, and related vectors, cells, compositions, methods and systems of the present disclosure that provide a bioengineering application of microcompartments that has not been previously explored.
[0303] In some embodiments, a bacterial cell obtainable by any one of the methods of the disclosure is described, and in particular a cell comprising at least one toxic non-native protein within at least one engineered microcompartment within the cell.
[0304] In some embodiments, a bacterial cell herein described further comprises various toxic non-native proteins wherein the various non-toxic proteins function and/or aggregate independently of or in combination with one another.
[0305] In some embodiments, in a bacterial cell herein described the various non-toxic proteins reside within the at least one microcompartment within the cell either independently of or in combination with one another.
[0306] In some embodiments, in a bacterial cell herein described various non-toxic proteins reside within at least two or more microcompartments within a cell.
[0307] In some embodiments, in bacterial a cell herein described at least one of the microcompartments comprises at least one additional component.
[0308] In some embodiments, in a cell herein described the at least one additional component is presented to the at least one toxic-nonnative protein.
[0309] In some embodiments, a composition is described comprising one or more bacterial cells obtained from any one of the methods of the disclosure, and/or by any one of the systems of the disclosure and/or any one of the cells herein described together with a suitable vehicle.
[0310] In some embodiments, the engineered microcompartment proteins, and related target proteins, insertion regions, tags. Linkers, engineered microcompartments, bacterial cells herein described can be comprised in a composition together with a suitable vehicle. The term "vehicle" as used herein indicates any of various media acting usually as solvents, carriers, binders or diluents for the non-native toxic proteins and/or related cells that are comprised in the composition as an active ingredient. In particular, the composition including the non-native toxic proteins and/or related cells can be used in one of the methods or systems herein described.
[0311] As disclosed herein, the engineered microcompartment proteins, the target proteins, insertion regions, tags, linkers regulatory sequences, vectors and/or related cells herein described can be provided as a part of systems to produce one or more non-native toxic proteins, and in particular can be used in methods to produce or provide a non-native toxic protein herein described. The systems can be provided in the form of kits of parts. In a kit of parts, the non-native toxic proteins, regulatory sequences, vectors and/or related cells and other reagents to produce or provide a non-native toxic protein can be comprised in the kit independently. The non-native toxic proteins, regulatory sequences, vectors and/or related cells can be included in one or more compositions, and each component can be in a composition together with a suitable vehicle.
[0312] Exemplary components of a kit of parts and of constructs herein described comprise the nucleotide sequence of the linkers, protease cleavage sites, and histidine affinity tags that are codon optimized for maximum expression in E. coli such as the one described in the Examples section. Additional components can include labeled molecules and in particular, labeled polynucleotides, labeled antibodies, labels, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure. The terms "label" and "labeled molecule" as used herein as a component of a complex or molecule referring to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term "fluorophore" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording "labeling signal" as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like.
[0313] In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the assay, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).
[0314] Further details concerning methods and system, cells and compositions of the present disclosure will become more apparent hereinafter from the following detailed disclosure of examples by way of illustration only with reference to an experimental section.
EXAMPLES
[0315] The engineered microcompartment proteins herein described and related engineered microcompartments, methods and systems for engineering bacterial cells, as well as bacterial cells herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
[0316] In particular, the following examples illustrate exemplary methods and systems for expressing non-native, cytotoxic proteins or pathways involving these proteins in engineered microcompartment proteins for synthetic biology applications. The following examples demonstrate that engineered microcompartment proteins are useful platforms to produce in a bacterial cell proteins that are toxic and/or degradable in the cell minimizing cytotoxicity and degradation while improve efficiency of non-native protein expression compared to other convention approach for protein expression. The development of this platform can be broadly used in various fields including biofuels, biopharmaceuticals, biodefense, bioremediation as well as many other applications in bioscience in general.
[0317] A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional non-native proteins and bacterial systems and related methods and systems according to embodiments of the present disclosure.
Example 1
Exemplary AMPs
[0318] Exemplary AMP used in some experiments herein described are listed in Table 1.
TABLE-US-00003 TABLE 1 Amino acid sequences of exemplary AMP AMP peptide Amino acid sequence SEQ ID NO HBCM2 KWKSFIKKLTKAAKKVVTTAKKPLIV 9 HBCM3 KWKKFIKSLTKSAAKTVVKTAKKPLIV 10 Cecropin PR- RRRPRPPYLPRPRPPPFFPPRLPPRIP 11 39 PGFPPRFPPRFP Apidaecin Ia GNNRPVYIPQPRPPHPRI 12
[0319] HBCM2 and HBCM3 are two AMPs that have activity against antibiotic-resistant P. aeruginosa. These two AMPs are optimized hybrid peptides of moth cecropin and bee melittin [18, 19]. These peptides also have been shown to have anti-inflammatory activity in cystic fibrosis mouse models [19]. Non-lytic AMPs cecropin PR-39 and apidaecin Ia, which are derived from pig and honeybee, respectively [25, 26] are also expressed. Collins and coworkers have shown that these two AMPs have high bactericidal activity but do not induce lysis of pathogens, avoiding release of endotoxins that may be toxic to the human host [27]. Thus, they may be better suited for therapeutic applications. All four of these AMPs (Table 1) lack cysteine residues and thus do not require disulfide bond formation for activity, making them more amenable to heterologous expression [28]. Other AMPs with activity toward P. aeruginosa based on the Antimicrobial Peptide Database can also be screened for expression [22].
Example 2
Encapsulation Strategies Using Encapsulins
[0320] Protein expression systems can be designed to express the AMPs encapsulated inside an encapsulin microcompartment system. In encapsulin systems (e.g., ferritin cages), a single, repeating encapsulin protein is responsible for encapsulating an interior protein, in a typical ratio of 60 encapsulins to 6 interior proteins [2, 29]. In contrast to other BMC systems, in which several different BMC shell proteins are required to encapsulate interior protein [30], encapsulin systems are less complex with a single encapsulating protein. They are therefore expected to have improved formation of compartments in a recombinant system, and thus higher loading of interior protein, compared to traditional BMC systems. These expression systems are initially designed under traditional inducible promoters (e.g., pTet, pT5) in order to test and optimize maximum production levels.
[0321] In order to target the AMPs to the interior of encapsulins, two potential strategies (FIGS. 9A-B) are employed. In the first strategy (FIG. 9A), the encapsulin shell protein will be directly fused to the C-terminus of the given AMP in one single polypeptide chain. The first strategy design is based on initial studies that suggest that interior protein-encapsulin fusions allow all interior proteins to be encapsulated by the encapsulin shells, without sacrificing the integrity of the formed encapsulin compartments. This fusion system is, furthermore, predicted to have more AMP molecules loaded into the encapsulin compartments (60:60 encapsulin:interior protein), compared to the native system (60:6 encapsulin:interior protein) [2], which should encourage higher production of AMPs in the recombinant system. The linker region between the interior protein and encapsulin can be optimized for efficient loading of AMPs into the encapsulins. Here, computational modeling can be performed to guide linker optimization for efficient loading into the compartments.
[0322] In some scenarios, the mechanism of action for AMP toxicity is carried out in the E. coli host before encapsulation occurs. This is a possibility since 60 units of the fusion protein will need to be synthesized to assemble one full encapsulin compartment, but only 1 unit of the fusion protein can cause toxicity. Thus, as an alternative to the first strategy, in the second strategy (FIG. 9B) a known C-terminal encapsulin targeting tag (18 amino acids, Encaptag) [2] will be fused to the AMP peptide and the AMP-Encaptag will be co-expressed with the encapsulin shell protein from two different promoter systems, pTet and pT5. In this system, the encapsulin shell proteins will be expressed first so that when the AMP-Encaptag protein is subsequently expressed, there will be sufficient encapsulin shell protein available for immediate AMP encapsulation. Toxicity shielding of AMPs and production amounts in either strategy will be determined using growth assays and SDS-PAGE/Western blots for quantification. If toxicity is shielded, better growth and higher production of AMPs can be expected in cells expressing AMP-encapsulin compared to AMP alone. Expression optimization will be performed to determine the maximum amount of encapsulated AMP that can be produced with limited toxicity to the host E. coli cells.
Example 3
Identification of Proteolysis Conditions
[0323] Once an expression system for encapsulating AMPs is established, conditions to release the AMPs from the interior of the encapsulins by proteolysis can then be developed. Here, proteolytical cleavage of AMPs from the encapsulin shell/Encaptag as well as cleavage of specific sites in the encapsulin shell protein can be applied so that the entire structure can be degraded for release of AMPS.
[0324] FIG. 11 illustrates the design of a protease-sensitive encapsulin. Antimicrobial peptide (AMP) is fused to the N-terminus of the encapsulin protein. Specific protease recognition sites are inserted between the AMP and encapsulin protein as well as within the encapsulin protein itself. The fusion protein is expressed from a T7 inducible promoter and purified. The purified fusion is digested with a specific protease that targets the protease recognition sites and the AMPS are released and thus can be isolated for a desired function.
[0325] Known protease cleavage sites (i.e., thrombin, enterokinase) [31] will be inserted between the AMP protein and encapsulin shell/Encaptag as well as within the encapsulin shell protein itself. Computation modeling will be used to identify potential cleavage insertion sites that would cause the encapsulin compartments to fall apart upon protease cleavage but would otherwise not affect the integrity of and loading into the compartments. Expression of AMP-encapsulin systems with appropriate protease cleavage sites will be tested for toxicity shielding and production amounts using growth assays and SDS-PAGE/Western blots. Microcompartments from stable AMP-encapsulin systems will be subsequently isolated using established differential centrifugation methods [2, 32] and treated with an appropriate protease to determine if AMP can be efficiently released from the compartments. Efficient release will be assessed by separation of AMP from encapsulin via size-exclusion chromatography or Ni-NTA affinity chromatography, where the AMP will be tagged with a His.sub.6 tag that will not be cleaved from the AMP during proteolysis. If release of AMP is efficient, the AMP and encapsulin can be expected to purify as separate fractions during chromatography, whereas if release is inefficient, co-purification of the components is expected.
[0326] Recognition sequences and cleavage sites of exemplary proteases are shown in
TABLE-US-00004 TABLE 2 / forward slash (/) indicates where protease cleaves the protein sequence. Table 2: Recognition sequences and cleavage sites of exemplary proteases Sequence Enzyme Name and Cleavage SEQ ID NO Human Rhinovirus (HRV) LEVLFQ/GP 13 3C Protease Enterokinase DDDDK/ 14 Factor Xa IEGR/ 15 Tobacco etch virus ENLYFQ/G 16 protease (TEV protease) Thrombin LVPR/GS 17
Example 4
Release of AMPs from E. coli
[0327] Once an appropriate protease-sensitive AMP-encapsulin system is identified, experiments can be performed to test whether AMP can be released from encapsulin within the E. coli host organism and subsequently from the cell itself by co-expression of the AMP-encapsulin system and the appropriate protease.
[0328] In these studies, the AMP-encapsulin system will be expressed first from a pTet (and pT5 if necessary) promoter. Once high levels of AMP-encapsulin are produced, protease expression will be induced from a different promoter (i.e., pT7 or pRha). A lysis protein, such as colicin E7 lysis protein [33] may also need to be co-expressed with the protease to achieve efficient lysis of the bacterial cell to release the AMPs. The kinetics of AMP release from cells into the spent medium over time upon proteolysis/lysis induction will be assessed via SDS-PAGE/Western blot.
[0329] In order to compare the lysis system to known systems [33, 34], control strains of E. coli that 1) secrete the AMP candidates via YebF secretion signals [35] and 2) produce AMPs without encapsulin, will be generated, which are released by lysis. The rate and total amounts of AMP released will be compared among the control strains and the protease-sensitive AMP-encapsulin strain in order to demonstrate improved AMP release in the latter system.
Example 5
Design of a Control System
[0330] The AMP-encapsulin and protease/lysis systems can be coupled to a specific pathogen, quorum-sensing system for P. aeruginosa. The quorum sensing system from P. aeruginosa was primarily chosen as a proof-of-principle because it is highly specific to P. aeruginosa, has been very well studied [36], and has already been adapted for synthetic biology applications [33, 34, 37]. P. aeruginosa has a unique N-acylhomoserine lactone (AHL) quorum sensing molecule, called 3-oxo-C12-homoserine lactone (3OC12HSL), that is specifically and directly sensed by the native transcription factor LasR. Binding of 3OC12HSL to LasR induces dimerization of LasR, which then is able to bind to and drive expression from pLas1 promoters [36].
[0331] The LasR1pLas1 system can be adapted to drive expression of the protease that specifically cleaves the encapsulins and if necessary, a lysis protein to lyse the E. coli Nissle 1917 host and ultimately release AMPS at high concentrations. Encapsulated AMPS can be expressed at high constitutive levels in order to ensure that they will be immediately released at high doses upon lysis. LasR can be expressed at medium constitutive levels to ensure that 3OC12HSL can be detected at any time (FIG. 10, panel A). Established, synthetic sigma70 promoters of different strengths can be used for constitutive expression [38] and these constructs will be inserted into the genome of the E. coli host to prevent variability in expression levels among cells.
[0332] In order to minimize premature proteolysis/lysis of the E. coli host, a negative feedback loop logic can be engineered into the protease/lysis cassette (FIG. 10, panel A). Here, the LasR transcription factor will drive expression of an ECF sigma factor (ECFsf) that is orthogonal to the other host E. coli sigma factors [39]. This sigma factor will then activate a unique ECFsf promoter to drive expression of the protease/lysis cassette as well as an anti-sigma factor. The anti-sigma factor will bind and inhibit the ECFsf to prevent further expression of the protease/lysis cassette. Thus, the protease/lysis cassette will only have sustained expression under high concentrations of 3OC12HSL, where the ECFsf is able to out-compete the anti-sigma factor. Alternatively, the anti-sigma factor can be constitutively expressed in order to create a threshold-gated switch, where a set concentration of ECFsf would need to accumulate to overcome the anti-sigma factor. Here, the expression levels of the anti-sigma factor would set the desired threshold. Constitutive expression of the anti-sigma factor also has the added benefit of reducing leaky expression from ECF promoters, which can also lead to premature lysis [39].
Example 6
Optimization and Testing of the System
[0333] Initial experiments can be conducted with fluorescent protein reporters (e.g., mVenus) [40] instead of the AMP-encapsulin and protease/lysis cassettes. With these reporters, the concentrations of 3OC12HSL needed to turn on different components of the circuit can be tested and these concentrations can be correlated to cell densities of P. aeruginosa [37]. Further optimization of this system can be performed to obtain the correct degree of expression at an appropriate 3OC12HSL concentration, which may involve altering any of the following: lifetimes of the components via ssrA degradation tags [41], translational production levels via changes in the ribosome binding site (RBS) [42], and copy number of the anti-sigma factor gene to enable sufficient inhibition of the ECFsf. Computational modeling of the rates of production and degradation of the system components will be performed to guide and improve design, according to current synthetic biology practices [43]. Then, the system can be tested using the actual AMP-encapsulin and protease/lysis gene cassettes. The amounts of AMP released into the medium over time can be measured using SDS-PAGE/Western blots as well as the degree of lysis using optical density at 600 nm and microscopy. These measurements can establish whether the system can efficiently deliver a high dose of AMPs. Experiments with P. aeruginosa and exogenously added AMPs can be conducted to determine if the amounts of AMPs released by the system are sufficient to kill the pathogen.
[0334] In some cases, the constitutive production of the AMP-encapsulins may be detrimental to the overall fitness of the host E. coli cell, either due to incomplete encapsulation or metabolic burden. In these cases, the system can be designed to only produce the AMP-encapsulin when 3OC12HSL is detected and subsequently lyse the cells after an appropriately spaced delay period at high 3OC12HSL levels (FIG. 10, panel B). The ECFsf downstream of the AMP-encapsulin can be expressed and the stability of the ECFsf (via ssrA tags) can be adjusted to only drive expression of the protease/lysis cassette at high 3OC12HSL levels, which may require further optimization.
Example 7
A Sense-Control-Release System for Clostridium difficile
[0335] A sense-control-release system for Clostridium difficile can also be developed based on recent studies identifying a putative, two-component, quorum-sensing system that can sense a unique C. difficile autoinducing peptide (AIP) and activate toxin production in the virulent form of C. difficile [44]. This system can replace the P. aeruginosa LasR sensing component in the sense-control-release system to specifically detect virulent C. difficile instead of P. aeruginosa and release a therapy in response. Because C. difficile is a gram-positive bacterium, the system need to be expressed in a gram-positive host, such as the probiotic gut organism, Lactococcus lactis, which has already been bioengineered for therapy applications [45, 46].
Example 8
Testing of Therapeutic Delivery System Efficacy In Bacterial Liquid Culture
[0336] The efficacy of the encapsulated AMP delivery system in killing P. aeruginosa can be tested. Initial studies are performed in bacterial liquid culture of planktonic P. aeruginosa cells (FIG. 12). Different concentrations of the therapeutic E. coli can be mixed with different concentrations of P. aeruginosa and the degree of killing of the P. aeruginosa can be measured by Bactolight live/dead staining (Thermo), OD600, and/or colony forming unit (CFU) counting over time. The E. coli can be differentiated from the P. aeruginosa in microscopy experiments by expressing a fluorescent protein marker in the E. coli. From these experiments, the number of E. coli cells required to kill a certain population of P. aeruginosa can be determined. As controls, two E. coli strains can be constructed that express the following in response to 3OC12HSL detection: 1) secreted AMPs and 2) non-encapsulated AMPs followed by lysis. The ability of these control strains to kill P. aeruginosa can be compared to that of the encapsulated AMP delivery system, in order to demonstrate improved killing in the latter system.
Example 9
Testing of Therapeutic Delivery System Efficacy In Biofilm Model
[0337] The ability of the therapeutic E. coli to kill biofilms of P. aeruginosa can also be tested (FIG. 13). Biofilms of P. aeruginosa are commonly found in infections [47]. Biofilms can be grown on pegs of polystyrene attached to a lid of a microtiter plate according to standard practices [48]. Biofilms can then be treated with different concentrations of the therapeutic E. coli by immersing the biofilm pegs in E. coli culture dispensed in the wells of a microtiter plate. The degree of biofilm dispersal can be assayed over time using standard crystal violet staining followed by absorbance measurements at 595 nm [49] as well as live/dead staining and/or CFU counting. Control strains described for the liquid culture can also be tested to demonstrate improved killing in our AMP-encapsulin strain.
Example 10
Engineered Therapeutic Cells for Improved Killing In Biofilm Model
[0338] AMPs have been shown to be more effective against bacterial biofilms compared to traditional small molecule antibiotics due their ability to act on non-growing cells as well as inhibit biofilm formation by preventing adhesion and the production of biofilm components (i.e., EPS) [50]. However, there is a possibility that the AMPs released by the E. coli are ineffective against the biofilm because they are not able to penetrate the EPS/DNA matrix to access the P. aeruginosa cells. In this case, a separate strain of E. coli can be engineered that will release DNasel or alginate lyase (AL) via lysis in response to P. aeruginosa detection in order to break up the biofilm. Both DNasel and AL have been shown to be effective in dispersing P. aeruginosa biofilms [51, 52]. The AMP-encapsulin gene cassette can be swapped for the genes for DNasel or AL to create the new strains. The DNaseI/AL strain and the original AMP-encapsulin strain can be mixed together and tested to determine if the combination treatment is more effective in killing P. aeruginosa using the methods previously described.
[0339] In order for the therapeutic E. coli to find and stay localized at the biofilm long enough for efficient therapeutic release, the E. coli can be engineered to chemotax toward the 3OC12HSL, using the method developed by Chang and coworkers [34], where LasR was designed to drive expression of cheZ, which promotes smooth swimming toward a metabolite of interest in a .DELTA.cheZ genetic background. Improved killing with the additional cheZ system can be demonstrated using the methods previously described.
Example 11
Testing of Therapeutic Delivery System Efficacy In Host Tissue Culture Model
[0340] The ability of our therapeutic E. coli to kill P. aeruginosa can also be tested in a host tissue culture model (FIG. 143). A co-culture of P. aeruginosa with intestinal epithelial cells can be treated with different concentrations of the therapeutic E. coli. In addition to assessing killing of the P. aeruginosa by live/dead staining and/or CFU counting, the fitness of the intestinal epithelial cells can also be tested by standard MTT assays [53] to determine if lysis of the E. coli or P. aeruginosa is detrimental to the host cells. The correlation between number of lysed cells and lower fitness of host cells, if any, can be quantified. If lysis of the E. coli results in significant loss of host cell fitness, several gene deletions in the E. coli strain can be made, which force the cells to only produce lipid IV.sub.A, instead of lipid A, the component of bacterial LPS that is responsible for endotoxic activity; lipid IV.sub.A, a precursor to lipid A has been shown to lack endotoxic activity [54]. While these deletions have been demonstrated to effectively reduce the endotoxic effect of E. coli [54], these deletions may reduce the fitness of the E. coli cells, which will need to be assessed to determine they have any effect on the use of our therapeutic E. coli.
Example 12
Growth and Expression of Constructs 124, 125, and 133 in C43(DE3) E. coli Cells
[0341] In this example, engineered microcompartment proteins are constructed and expressed in C43 (DE3) E. coli cells.
[0342] As shown in FIGS. 14A-C, the gene sequence for Apidaecin Ia peptide (Ap) was fused to the 3' end of gene sequences for various encapsulin (Encap) constructs. The gene fusions were placed under the control of a T7 promoter in the commercially available pET24a vector. These gene fusions include those that express the following proteins: 1) Ap fused to Encapsulin containing a TEV protease cleavage site and His-tag after position K138 (Ap-Encap(K138) from pMCY124) (FIG. 15A); 2) Ap fused to Encapsulin containing a TEV protease site after position K71 and the TEV protease site and His-tag after position K138 (Ap-Encap(K71,K138) from pMCY125) (FIG. 15B); and 3) Ap fused to Encapsulin containing a TEV protease site after position D60 and the TEV protease site and His-tag after position K138 (Ap-Encap(D60,K138) from pMCY133) (FIG. 15C).
[0343] In the constructs herein described, a linker comprised of a Tobacco Etch Virus (TEV) protease site (sequence: ENLYFQG) followed by a GTS (Gly-Thr-Ser) linker is placed between the Ap peptide and encapsulin monomer, in order to enable later cleavage of Ap from encapsulin via TEV protease.
[0344] In constructs pMCY125 and pMCY133, within the encapsulin monomer, two specific protease sites are also inserted to enable digestion of the encapsulin cage.
[0345] In pMCY125, the first site is inserted following amino acid residue K71 in the encapsulin monomer. This site is chosen because of its location between the surface-accessible E-loop and P-domain of the encapsulin structure. Cleavage at this location was predicted to disrupt the structure of the encapsulin cage. A TEV protease site surrounded by double Gly (Gly-Gly) linkers on both sides of the site is inserted following residue K71. The second site was inserted following amino acid residue K138. This site was shown to be surface-accessible and insertion of a His.sub.6-tag at this location allowed for Ni-NTA affinity purification of the encapsulin cage [55]. A TEV protease site was inserted directly adjacent to the His.sub.6-tag on the N-terminal side. Thus, the following sequence was inserted following residue K138: a pentaglycine linker (Gly.sub.n), a TEV protease site, a His.sub.6-tag, and another pentaglycine linker. The His.sub.6-tag was used for affinity purification of the Ap-EncapK71K138 construct. The codon-optimized nucleotide sequence for pMCY125 (SEQ ID NO: 18) is also shown in FIG. 15A.
[0346] As controls, gene fusions expressing the following proteins were also prepared: 1) Ap fused to a TEV protease cleavage site alone (Ap-TEV from pMCY126); 2) Ap fused to thioredoxin (Ap-Trx from pMCY117); and 3) Ap fused to the C-terminus of Encapsulin containing the TEV protease site and His-tag after position K138 (Encap(K138)-Ap from pMCY123--Here, Ap peptide would be outward facing in the Encapsin compartment, making it potentially toxic and susceptible to proteolysis.) All gene constructs are diagramed in FIG. 16. The peptide Apidaecin Ia has a sequence of SEQ ID NO: 19: GNNRPVYIPQPRPPHPRIENLYFQ
[0347] All DNA plasmids containing the gene constructs were transformed into C43(DE3) E. coli cells (Lucigen) and expression of the corresponding protein constructs were tested. Cells were grown in 10 mL of Luria-Bertani (LB) medium in 50-mL flasks to mid-log phase (optical density at 600 nm (OD600) of 0.4). Isopropyl f3-D-thiogalactoside (IPTG) was then added to the culture to a final concentration of 0.1 mM and culture was grown for another 4 h at 30.degree. C. to induce protein expression. After induction, cells were harvested and resuspended in 400 uL of lysis buffer containing 60% BPER-II detergent (Thermo-Fisher) in buffer A (50 mM Tris pH8.0, 500 mM KC1, 12.5 mM MgCl.sub.2) supplemented with 0.1 mg/mL lysozyme and 10 U/mL of DNaseI. Cells were lysed by incubating at 4.degree. C. with occasional mixing for 15 min. Cells were then centrifuged at 12,000 g, 4.degree. C. for 25 min and the supernatant was collected as the soluble fraction. Samples prior to centrifugation were saved as the "whole" samples, while the supernatant after centrifugation was saved as the "soluble" samples.
[0348] All samples were resolved on an any-kDa SDS-PAGE gel (Bio-rad) and stained with Coomassie blue (FIG. 17). Arrows denote predicted location of the expressed protein. Samples expressing fusions with Ap on the N-terminus of Encapsulin are highlighted within the box. Only these fusions show high expression of soluble, induced protein (only soluble protein is available for purification). All control samples do not have detectable expression of the given protein construct.
Example 13
Purification of Constructs 124, 125, and 133
[0349] Purification of the protein constructs Ap-Encap(K138), Ap-Encap(K71,K138), and Ap-Encap(D60, K138) described in Example 12 is performed in this example.
[0350] Protein was purified using Ni-NTA affinity resin that binds to the His-tag on the Encapsulin constructs. Cells from 50 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) supplemented with 10 mM imidazole and 10 U/mL of DNaseI. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4.degree. C. for 25 min. The supernatant (soluble) was isolated, added to 250 uL of Ni-NTA resin (Qiagen), and equilibrated with the resin at 4.degree. C. for 45 min with rocking. The resin was then packed into a column and the flow through (FT) was collected. The resin was then washed with 5.times.1 mL fraction of 20 mM imidazole in phosphate buffer. Protein was eluted with 7.times.200 uL fractions of 250 mM imidazole in phosphate buffer (fractions E1-E7). Fractions E2-E7 were pooled for each sample, concentrated, dialyzed into 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.
[0351] For each purification, samples of the soluble fraction before purification, the flow through, and elution fractions E1-E7 were resolved on an any-kDa SDS-PAGE and stained with Coomassie blue (FIG. 18).
[0352] Based on the gel, only the Ap-Encap(K71,K138) protein bound to the Ni-NTA resin to high affinity and was thus subsequently purified to high purity (-99% pure). The other constructs did not bind well to the column and thus could not be purified to high purity. These results suggest that the Ap-Encap(K71,K138) protein may have an altered structure compared to the other two proteins, allowing for greater accessibility of the His-tag for purification.
[0353] Final yields from the purification are shown in Table 3.
TABLE-US-00005 TABLE 3 Yields from the purification for constructs 124, 125 and 133 Theoret. Ap mg/ total total L mg/L mg/L Construct mL uL mg culture culture culture 124 = Ap- 1.02 300 0.307 0.05 6.1 0.51 Encap(K138) 125 = Ap- 5.40 280 1.51 0.05 30.3 2.5 Encap(K71, K138) 133 = Ap- 1.38 260 0.359 0.05 7.2 0.58 Encap(D60, K138)
[0354] In Table 3, the columns from left to right show: construct name, concentration of the purified protein in mg/mL, total volume in uL, total amount in mg, total volume of culture that the protein originated from in L, yield of protein in mg of protein per L of original culture, and theoretical yield of the Ap peptide after protease cleavage assuming 100% proteolysis in mg of Ap peptide per L of original culture. The Ap-Encap(K71,K138) protein clearly has the highest yield, nearly 5 times higher than the other proteins.
Example 14
TEV Protease Cleavage of the Purified AP-Encapsulin Fusions
[0355] In this example, experiments were conducted to show TEV protease cleavage of the purified Ap-Encapsulin fusions.
[0356] 60 ug of purified material was digested with 30 U of TEV protease enzyme in a 80 uL reaction with 1 mM DTT and lx TEV protease buffer from Promega. Aliquots of digested material were removed after 1 h, 3 h, and 20 h of digestion at 30 oC. A sample without TEV protease was also prepared as the 0 h sample.
[0357] All samples were analyzed by SDS-PAGE using both an any-kDa gel to analyze fragment >15 kDa (FIG. 19A) and a 16.5% Tris-Tricine gel to analyze fragments <15 kDa (FIG. 19B). Samples were also analyzed by Western blot using an anti-TEV site antibody (FIG. 19C). Arrows on the gels above show digested fragments.
[0358] P (light gray arrow) denotes TEV protease. U (light gray arrow) denotes undigested material. Underlined numbers (white arrows) denote partially digested material containing Ap peptide. Italicized numbers (dark gray arrows) denote partially digested material NOT containing Ap peptide. Bold numbers (black arrows) denote fully digested material. Table 4 below shows the predicted protein fragments expected from digestion. The numbers are the expected fragment sizes in kDa. The numbers in parentheses correspond to the numbers shown on the gels and Western blot.
TABLE-US-00006 TABLE 4 Predicted protein fragments from digestion Degree of digestion Construct 124 125 133 Undigested Ap-Encap1-Encap2- 36.2 37.2 37.3 Encap3 partial Encap1-Encap2- 34.2 (5) 34.3 digestion Encap3 Ap-Encap1-Encap2- 21.4 (6) 21.5 (12) TEV Encap2-Encap3 24.7 26.1 Encap1-Encap2-TEV 33.2 18.4 (7) 18.5 (13) Ap-Encap1-TEV or 20.4 (1) 12.5 11.2 Ap-Encap2-TEV full Encap3 15.8 (3) 15.8 (8) 15.8 (14) digestion Encap2-TEV 17.4 (2) 8.9 (10) 10.3 (15) Encap1-TEV 9.5 (9) 8.2 Ap-TEV 3.0 3.0 (11) 3.0
[0359] The data of Table 4 show that construct 125 (Ap-Encap(K71, K138)) is digested by TEV protease to near completion, which is most clearly observed by the disappearance of undigested material (U) and band (6) denoted by the white arrow and the appearance of the Ap peptide band (11) after 20 h digestion. The other constructs do not have clear disappearance of undigested bands denoted by the white arrows based on Western blot, suggesting that they are not well digested by TEV protease. It is also noted that the amount of construct 125 is much higher than the other 2 constructs. Therefore, in the Western blot, the proportion of the band denoted by the white arrow relative to the original is much lower than the other constructs, suggesting nearly complete digestion.
Example 15
Expression of Tandem Ap Peptides Fused to Encapsulin in C43 (DE3) E. coli Cells
[0360] This example shows expression of tandem Ap peptides fused to the Encapsulin construct with insertions after K71 and K138.
[0361] Gene cassettes expressing up to 4 Ap peptides fused to a single Encapsulin construct were made (FIG. 20A). TEV protease cleavage sites were placed between each Ap peptide as well as prior to the first Ap peptide and prior to the Encapsulin construct in order to ensure the same Ap peptide sequence is obtained upon full protease digestion. The sizes of the tandem Ap peptides that were fused to the Encapsulin construct are as follows: 1xAp=32 amino acids; 2xAp=57 amino acids; 3xAp=82 amino acids; 4xAp=107 amino acids. The gene cassettes were placed under the control of a T7 promoter in the commercially available pET24a. The DNA plasmids were transformed in C43(DE3) E. coli cells and expressed under the same conditions as described above. Cells were lysed and soluble fractions were also obtained in the same manner as described above.
[0362] All "whole" and "soluble" samples were resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue (FIG. 20B). Arrows denote predicted location of the expressed protein. Only 1xAp and 2xAp peptide fused to Encapsulin could be expressed. The 3xAp and 4xAp peptide-Encapsulin fusions were not detectable by SDS-PAGE. These data suggest that up to 57-amino acid peptides could be fused to Encapsulin in order to achieve expression. An 82-amino acid peptide fused to Encapsulin could not be expressed, suggesting this fusion and fusions with larger peptide attachments can not properly form Encapsulin microcompartments in order to achieve expression.
Example 16
Expression of Tandem HBCM-2 (HB) Peptides Fused to Encapsulin In C43(DE3) E. coli Cells
[0363] This example describes the expression of tandem HBCM-2 (HB) peptides fused to the Encapsulin construct with insertions after K71 and K138. The HBCM-2 peptide has a sequence of SEQ ID NO: 9 from Table 1.
[0364] Gene cassettes expressing up to 3 HB peptides fused to a single Encapsulin construct were made as well as a control of HB fused to thioredoxin (Trx) (FIG. 21A). The sizes of the tandem HB peptides that were fused to the Encapsulin construct are as follows: 1xHB=33 amino acids; 2xHB=66 amino acids; 3xHB=99 amino acids. The gene cassettes were placed under the control of a T7 promoter in the commercially available pET24a. The DNA plasmids were transformed in C43(DE3) E. coli cells and expressed under the same conditions as described above. Cells were lysed and soluble fractions were also obtained in the same manner as described above.
[0365] All "whole" and "soluble" samples were resolved on an any-kDa SDS-PAGE gel and stained with Coomassie blue (FIG. 21B). Arrows denote predicted location of the expressed protein. Only 1xHB and 2xHB peptide fused to Encapsulin could be expressed. The 3xHB peptide-Encapsulin and HB-Trx fusions were not detectable by SDS-PAGE. These data suggest that up to 66-amino acid peptides could be fused to Encapsulin in order to achieve expression. A 99-amino acid peptide fused to Encapsulin could not be expressed, suggesting this fusion and fusions with larger peptide attachments cannot properly form Encapsulin microcompartments in order to achieve expression.
[0366] These data confirm the observation at HB can only be expressed when fused to Encapsulin, but not to Trx, demonstrating that only Encapsulin has a toxicity/proteolysis shielding effect that allows HB to be expressed.
Example 17
Design of HB-Enc Constructs with TEV Protease Cleavage Site Insertions
[0367] HB-Enc constructs with TEV protease cleavage site insertions were designed based on the following approach.
[0368] To express HBCM2 within the lumen of an encapsulin cage, the peptide was directly fused to the N-terminus of the Enc monomer, which is luminal-facing based on the X-ray crystal structure of the encapsulin cage [56]. This strategy was chosen to maximize incorporation of expressed peptide into the Enc cage. Targeting sequences that associate with the lumen of the Enc cage have been identified for loading interior protein [56, 57]. However, quantitative loading using targeting tags in this and other protein compartment systems is often incomplete in heterologous systems and remains a significant challenge [58] [57, 59-61] Direct fusion is expected to ensure each peptide is associated with Enc monomer to maximize loading into the Enc cage.
[0369] To isolate HBCM2 following purification of the Enc cage, TEV protease recognition sites were placed between the peptide and the Enc monomer as well as at several surface accessible locations to encourage cage disassembly (FIGS. 22A-B). Kang and coworkers [55] previously demonstrated that a His.sub.6-tag could be placed after exterior residue K138 (EncK138.sup.His) and was sufficiently surface exposed for purification via Ni-NTA chromatography. Thus, a construct, HB-EncK138.sup.TEV-His, containing the following features was initially designed: 1) HBCM2, a TEV site, followed by a G.sub.4T-linker fused to the N-terminus of Enc; and 2) a TEV site followed by a His.sub.6-tag inserted after residue K138 with G.sub.5-linkers flanking both ends of the insert.
[0370] Additional sites within the Enc monomer were also identified, which would be surface accessible and amenable to insertion with minimal disruption to the cage structure by examining the conservation of each residue and its flexibility (RMSFA2) based on a reported crystal structure PDB: 3DKT. Residues within the loop of the E-domain of Enc were found to be surface accessible with the highest degree of flexibility and relatively low conservation. Residues D60 and V57 were chosen for insertion because they are at the middle and start of the loop, respectively. Additionally, K71 was also chosen for insertion because its location at the end of the E-domain, immediately preceding the P-domain was thought to hold promise for cage disassembly; the residue is exposed to the exterior and has minimal conservation.
[0371] Thus, HB-Enc fusions were constructed with a TEV site flanked with G.sub.2-linkers following residues D60, V57 or K71, in addition to the TEV-His insertion following residue K138. These constructs are referred to as HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEV-His, and HB-EncK71.sup.TEVK138.sup.TEV-His, respectively (FIG. 22A).
Example 18
Expression of HB Peptide Fused to Different Engineered Encapsulin (Enc) Constructs Compared to HB Peptide Fused to Other Common Carrier Proteins In C43(DE3) E. coli
[0372] The constructs comprising HB peptide designed as indicated in Example 17 were tested in comparison with constructs where HB peptide is fused to other common carrier proteins. In particular, expression of N-terminal HB-fusions to the following other common carrier proteins with C-terminal His-tags was also tested: Small ubiquitin-like modifier (SUMO), thioredoxin (Trx), glutathione S-transferase (GST), and maltose-binding protein (MBP).
[0373] The amino acid sequences of the tested constructs are shown in FIGS. 22B.
[0374] In particular, in order to perform the testing, all DNA plasmids were transformed and expressed in C43(DE3) cells as described in Example 12, except that cells were grown in 25 mL of LB medium in 150-mL flasks. IPTG induction was conducted overnight for 17 h at 18.degree. C. to enhance soluble protein expression. Cells were harvested and lysed also as described in Example 12.
[0375] All N-terminal HB-Enc constructs showed over-expression of soluble HB-Enc protein by SDS-PAGE and anti-His-tag Western blot (FIG. 23A). In contrast, a C-terminal Enc-HB fusion was not expressed as soluble protein. The HB-EncK71.sup.TEVK138.sup.TEV-His construct showed the most robust expression with the highest cell density (OD.sub.600 of 5.8) (FIG. 23B), compared to the other double TEV site containing constructs (OD.sub.600 of 3.7-4.0). Removal of the K138.sup.TEV-His site from this construct to produce HB-EncK71.sup.TEV maintained this higher level of expression and cell density (OD.sub.600 of 5.6), which was also similar to the EncK138.sup.His control.
[0376] Expression of N-terminal HB-fusions to the following other common carrier proteins with C-terminal His-tags was also tested: Small ubiquitin-like modifier (SUMO), thioredoxin (Trx), glutathione S-transferase (GST), and maltose-binding protein (MBP) (FIG. 23B). These constructs had minimal expression in C43(DE3) cells. Only HB-SUMO could be detected by SDS-PAGE, while all other constructs were only detected by anti-His-tag Western blot.
[0377] Expression of HB-Trx, HB-GST, and HB-MBP were found to produce truncated products, demonstrating that they are sensitive to proteolysis in cell lysate. Given that the truncated products can be detected by anti-His-tag and are nearly full length (<5 kDa difference) indicates that proteolysis occurred on the N-terminus of the protein close to or within the HBCM2 sequence. Expression of EIB-EncK71.sup.TEVK138.sup.TEV-His also showed some truncated products by Western blot, but degradation was minimal compared to HB-Trx, HB-GST, and HB-MBP. All other HB-Enc fusions did not show truncated products. HB-EncK71.sup.TEV cannot be detected by anti-His-tag given its lack of a His-tag.
[0378] The above results indicate that HB-Enc fusions exhibit robust expression in E. coli C43(DE3) cells compared to other HB-carrier protein fusions
[0379] In particular, the expression data herein described reveals that HB peptide requires fusion to Enc for robust expression in C43(DE3) cells
Example 19
Purification and TEV Protease Digestion of HB-Enc Fusions
[0380] Constructs HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEV-His were purified as follows. Cells from 50 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) and 10 U/mL of DNasel. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4.degree. C. for 25 min. The supernatant (soluble) was isolated and then heated at 85.degree. C. for 15 min. The material was then centrifuged at 12,000 g, 4.degree. C. for 10 min and the soluble fraction was collected. Constructs were further purified by addition of ammonium sulfate to 25%(w/v) followed by centrifugation at 12,000 g, 4.degree. C. for 25 min to collect the insoluble pellet. Purified material was re-suspended in 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.
[0381] Construct HB-EncK71.sup.TEV was purified as follows. Cells from 50 mL of culture were harvested and resuspended in 60% B-PER II in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl), supplemented with 0.1 mg/mL lysozyme and 10 U/mL DNase. Cell lysate was incubated at 4.degree. C. for 1 h to lyse the cells. The supernatant (soluble) was isolated and then heated at 70.degree. C. for 15 min. The material was then centrifuged at 12,000 g, 4.degree. C. for 10 min and the soluble fraction was collected. The soluble fraction was dialyzed overnight at 4.degree. C. into phosphate buffer to remove the B-PER. The material was further purified by addition of ammonium sulfate to 50%(w/v) followed by centrifugation at 12,000 g, 4.degree. C. for 25 min to collect the insoluble pellet. Purified material was re-suspended in 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.
[0382] Construct HB-EncK71.sup.TEVK138.sup.TEV-His was partially purified Ni-NTA chromatography. Cells from 50 mL of culture were harvested and resuspended in 60% B-PER II in phosphate buffer (25 mM sodium phosphate pH 7.5, 100 mM NaCl), supplemented with 0.1 mg/mL lysozyme and 10 U/mL DNase. Cell lysate was incubated at 4.degree. C. for 1 h to lyse the cells. The supernatant (soluble) was isolated, added to 250 uL of Ni-NTA resin (Qiagen), and equilibrated with the resin at 4.degree. C. for 45 min with rocking. The resin was then packed into a column and the flow through (FT) was collected. The resin was then washed with 5.times.1 mL fraction of 5 mM imidazole in phosphate buffer. Protein was eluted with 7.times.200 uL fractions of 250 mM imidazole in phosphate buffer (fractions E1-E7). Fractions E2-E7 were pooled for each sample, concentrated, dialyzed into 25 mM sodium phosphate pH 7.5, 100 mM NaCl, and stored for analysis.
[0383] B-PER II was found to at least partially prevent proteolysis of HB-EncK71.sup.TEV and HB-EncK71.sup.TEVK138.sup.TEV-His, whereas many different protease inhibitors including pepstatin, EDTA, PMSF and Roche protease inhibitor cocktail were not effective.
[0384] All purified constructs were digested by addition of exogenous TEV protease, followed by overnight incubation at 4.degree. C. Products were resolved on a 16.5% Tris-Tricine gel (Bio-Rad). All constructs were found to at least partially release HBCM2 peptide (FIG. 24). HB-EncK71.sup.TEVK138.sup.TEV-His and HB-EncK71.sup.TEV released the most peptide with apparent complete digestion based on the sizes of the fragments observed after digestion. In contrast based on densitometry analysis, HB-EncK138.sup.TEV-His, EncD60.sup.TEVK138.sup.TEV-His, and HB-EncV57.sup.TEVK138.sup.TEV-His released fewer peptide per fusion protein. These constructs had a relative HB/HB-Enc ratio of 0.26 to 0.56, assuming that the HB/HB-EncK71.sup.TEVK138.sup.TEV-His is 1.
[0385] HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEN-His constructs with a GT linker between the TEV site and the N-terminus of Enc, instead of the flexible G.sub.4T linker, were also purified and tested for digestion for TEV protease digestion. However, the HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, and HB-EncV57.sup.TEVK138.sup.TEV-His with the shorter GT linker did not result in detectable release of HB peptide by SDS-PAGE (FIG. 25). These results suggest that the G.sub.4T linker is crucial to release of peptide for these constructs.
Example 20
Insertion of TEV Protease Site at Position K71 Disrupts Cage Formation Which Enables Highly Efficient Release of Peptide by TEV Protease
[0386] K71.sup.TEV containing constructs as well as its susceptibility to proteolysis, all of the HB-Enc constructs were analyzed by size-exclusion chromatography (SEC) and native PAGE to test for cage formation (FIGS. 33A and 33B). SEC analysis revealed that HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEV-His, and the control EncK138.sup.His constructs primarily contain a high molecular weight (MW) species that migrated close to void volume, indicative of cages. In contrast, the majority of HB-EncK71.sup.TEVK138.sup.TEV-His migrated as lower MW species. HB-EncK71.sup.TEV appeared to contain some high MW species, but primarily consisted of the lower MW species. These results were confirmed by Native PAGE which can also be used to test for cage formation. Protein is resolved by native PAGE on an any-kDa gel (Bio-Rad) in the absence of SDS in the running and loading buffers.
[0387] All purified HB-Enc constructs (with G.sub.4T linker) were analyzed by native PAGE (FIG. 26). Native PAGE analysis revealed that HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncV57.sup.TEVK138.sup.TEV-His, and the control EncK138.sup.His constructs primarily contain a high molecular weight (MW) species that did not enter the gel, which is indicative of cages. In contrast, the majority of HB-EncK71.sup.TEVK138.sup.TEV-His migrated as lower MW species that entered the gel. HB-EncK71.sup.TEV appeared to contain some high MW species, but primarily consisted of the lower MW species.
[0388] The high and low MW species can be isolated by size exclusion chromatography (SEC) on a SHIMADZU FPLC with a Superose 6 increase 3.2/300 column. Typically, 50 .mu.L of a 1 mg/mL protein solution was loaded onto the column. Protein was eluted with 50 mM NaH.sub.2PO.sub.4, pH 8.0, 200 mM NaCl at a flow rate was 0.2 mL/min for 20 min. Species migrating at a retention time of 7-8 min were collected as the high MW species. Species migrating at a retention time of 10-11 min were collected as the low MW species. Example SEC traces for Ap-EncK138.sup.TEV-His and Ap-EncK71.sup.TEVK138.sup.TEV-His can be found in FIG. 27.
[0389] Species can be analyzed by transmission electron microscopy (TEM). Purified material, 10 .mu.L at a concentration of .about.0.1-0.25 mg/mL, was spotted on a Cu grid coated with carbon type B-formvar for 3 min. Material was wicked away using filter paper. Grid was washed once with 10 .mu.L of water for 1 min, then wicked away. Grid was then stained with 2% uranyl acetate in water for 3 min. Stain was wicked away and grid was washed an additional two more times with 10 .mu.L of water for 1 min each wash. Grids were dried at room temperature for at least 1 h prior to TEM. Grids were imaged on a FEI Titan TEM at 80 kV.
[0390] Isolation of the high and low MW HB-Enc constructs after SEC followed by transmission electron microscopy (TEM) analysis revealed that the high MW species indeed were comprised of encapsulin cages, while no cages were observed for the low MW species (FIG. 28). All observed cages were .about.25 nm in diameter, with no significant difference observed among the different HB-Enc constructs.
[0391] Similar results for Native PAGE and TEM analyses were obtained for Ap-Enc fusions (FIG. 29).
Example 21
Cage Forming HB-Enc Constructs are Resistant to Proteolysis In Cell Lysate
[0392] To test for proteolysis in cell lysate, cells expressing the HB-Enc constructs from 25 mL of culture were harvested and resuspended in phosphate buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl) and 10 U/mL of DNaseI. Cells were lysed via a French Pressure cell at 14,000 psi and then centrifuged at 12,000 g, 4.degree. C. for 25 min. The supernatant (soluble) fraction was collected and incubated at 4.degree. C. overnight. Samples for SDS-PAGE analysis were collected immediately after lysis and after overnight incubation.
[0393] As controls, samples after BPER-II lysis were also prepared. Samples were resolved on an any-kDa SDS-PAGE gel (Bio-Rad) and stained with Coomassie blue. For Western blot analysis, samples were resolved on an any-kDa and blotted to a PVDF membrane using a Transblot Turbo System (Bio-Rad) at 2.5 A for 7 min. Samples were probed using a mouse anti-His-tag primary antibody and a rabbit anti-Mouse-HRP conjugated secondary antibody (both from Bio-Rad). HRP was detected on the membrane using chemiluminescence.
[0394] All three cage-forming constructs (HB-EncK138.sup.TEV-His, HB-EncD60.sup.TEVK138.sup.TEV-His, and HB-EncV57.sup.TEVK138.sup.TEV-His) were resistant to proteolysis (FIG. 30). Full length product was observed even after overnight incubation with minimal degradation products. In contrast, non-cage forming HB-EncK71.sup.TEVK138.sup.TEV-His and HB-EncK71.sup.TEV were both significantly degraded immediately after French press lysis and completely proteolyzed after overnight incubation. Given the detection of degradation products by anti-His-tag, proteolysis appears to primarily occur at the N-terminus of the HB-Enc fusion, degrading the HB peptide.
[0395] These results show that cage-forming HB-Enc constructs are capable of protecting the HB peptide from proteolysis, which has also been observed in other protein compartment systems [62]. In contrast, the non-cage forming, K71.sup.TEV containing HB-Enc constructs are susceptible to proteolysis, which very likely accounts for their ability to be completely digested by TEV protease. Interestingly, these constructs are able to be highly over-expressed in C43(DE3) E. coli compared to other HB-carrier protein fusions, suggesting that the K71.sup.TEV containing Enc constructs still enable HB expression, perhaps by partial occlusion of the peptide to prevent it from carrying out its toxic mode of action.
Example 22
Released HB-TEV Peptide has Anti-Bacterial Activity Against E. Coli, While Ap-TEV Peptide is Inactive
[0396] The anti-bacterial activity of HB-EncK71.sup.TEVK138.sup.TEV-His and TEV protease-digested HB-EncK71.sup.TEVK138.sup.TEV-His were tested for growth inhibition activity against BL21(DE3) E. coli cells
[0397] Growth inhibition activity can be tested against E. coli BL21(DE3) cells. Overnight cultures of cells were diluted to 200 .mu.L of an initial OD.sub.600 of 0.05-0.1 in LB medium. Diluted cultures were grown at 37.degree. C., 1000 rpm in a PHMP-4 Microplate Shaker (Grant Instruments, Cambridge, England). Protein was added to the culture at an OD.sub.600 of 0.1-0.2. Growth measured by OD.sub.600 was monitored over time and were conducted in triplicate. TEV-digested material contained HB-Enc fusion protein (0.5 mg/mL) that was incubated with TEV protease (0.5 U/mL) overnight for 17 h at 4.degree. C.
[0398] The results are shown in FIG. 31, panel A. The HB-EncK71.sup.TEVK138.sup.TEV-His fusion protein did not inhibit the growth of E. coli at a concentration of 40 .mu.g/mL. However, TEV protease-digested EncK71.sup.TEVK138.sup.TEV-His showed significant bacteriostatic activity (theoretical HB peptide released was 4 .mu.g/mL). A control with the addition of TEV protease alone also showed no growth inhibition, confirming that the inhibitory activity was due to released HB peptide.
[0399] Growth inhibition assays of chemically synthesized M-HB-TEV peptide (M-HBCM2-TEV, the peptide product that is released by digestion) showed significant anti-bacterial activity at 2 .mu.g/mL (FIG. 31, panel B), consistent with the reported MIC [63], suggesting that the N-terminal Met residue and the residual C-terminal TEV site did not significantly affect activity.
[0400] Growth inhibition assays of chemically synthesized native Ap peptide and M-Ap-TEV peptide only showed significant anti-bacterial activity for the native Ap peptide starting at 100 .mu.g/mL. The M-Ap-TEV peptide as inactive against BL21(DE3) E. coli. Thus, the Ap-Enc system serves as a model/case study for a non-active peptide.
Example 23
Expression of HB-Enc Constructs in TOP10 E. coli Cells from an Arabinose Inducible System
[0401] All HB-Enc constructs as well as HB-Trx, HB-SUMO, HB-GST, and HB-MBP were cloned under a pBAD promoter for expression in TOP10 E. coli. Expression in TOP10 cells under 40 .mu.M (low) or 10 mM (high) arabinose induction was tested. Overnight cultures of cells were diluted to 200 .mu.L of an initial OD.sub.600 of 0.1 in LB medium. Diluted cultures were grown at 37.degree. C., 1000 rpm in a PHMP-4 Microplate Shaker (Grant Instruments, Cambridge, England). Arabinose inducer was added to the indicated concentrations once cultures reached an OD.sub.600 of 0.2. After 4-5 h induction, cells were harvested and lysed as described in [00254] and tested for expression by SDS-PAGE analysis and anti-His.sub.6 Western blot.
[0402] HB-Trx, HB-SUMO, and HB-GST had little to no detectable expression by SDS-PAGE analysis in TOP10 E. coli cells under an arabinose-inducible pBAD promoter (FIG. 33A). A partial HB-MBP was expressed under high arabinose induction (10 mM) but was truncated without the HB peptide at the N-terminus (FIG. 33B). In contrast, HB-EncD60.sup.TEVK138.sup.TEV-His, HB-EncK71.sup.TEVK138.sup.TEV-His, and HB-EncK71.sup.TEV could all be well expressed in the system under low arabinose induction (40 .mu.M) conditions. Some truncation of the K71.sup.TEV containing constructs was observed in the expression gel, but it was not as significant as truncation of HB-MBP.
[0403] Despite low expression of HB-Trx, HB-SUMO, and HB-GST and truncated expression of HB-MBP under 10 mM arabinose induction, cells expressing these constructs reached a significantly lower OD.sub.600 after induction (1.9-2.4) compared to cells significantly over-expressing HB-EncK71.sup.TEVK138.sup.TEV-His and HB-EncK71.sup.TEV constructs at 40 .mu.M arabinose (OD.sub.600 2.7) (FIG. 33C). These results suggest that the HB-Trx/SUMO/GST/MBP constructs may be conferring some toxicity to the expression cells, whereas the K71.sup.TEV containing HB-Enc constructs confer limited toxicity even when highly over-expressed. Expression of HB-EncD60.sup.TEVK138.sup.TEV-His consistently results in lower cell density (OD.sub.600 2.3) compared to expression of the K71.sup.TEV containing HB-Enc fusions, likely associated with some insoluble cage expression.
[0404] Overall, these results are similar to the C43(DE3)/T7 induction system, where fusion of HB to the encapsulin constructs enabled its expression and prevented its proteolysis during expression.
Example 24
Expression of Protease-Sensitive HB-Trx, HB-SUMO, HB-GST, and HB-MBP Constructs in BL21(DE3) E. coli Cells from a T7 IPTG Inducible System
[0405] HB-Trx, HB-SUMO, HB-GST, and HB-MBP were expressed in BL21(DE3) from a T7 IPTG inducible promoter using the same method as for C43(DE3) cells described in Example 12. Protease sensitivity of the constructs in lysate were tested as described in Example 21.
[0406] HB-Trx, HB-SUMO, HB-GST, and HB-MBP were found to be significantly over-expressed in BL21(DE3) E. coli (FIG. 34A).
[0407] Despite significant over-expression of these constructs, they are all highly susceptible to proteolysis after cell lysis (FIG. 34B). Immediately following lysis in the presence of B-PER-II, significant proteolysis of HB-GST and HB-MBP were observed by SDS-PAGE, contrasting with the HB-Enc constructs which did not exhibit significant proteolysis in the presence of B-PER (FIG. 30). Following French press lysis in the absence of B-PER, all other HB carrier protein fusions were subjected to rapid proteolysis with complete disappearance of full-length protein after overnight incubation at 4.degree. C.
[0408] It is possible that expression of non-Enc HB carrier protein fusions in BL21(DE3) cells is due to rapid protein synthesis relative to the rate of proteolysis in this strain, in contrast to C43(DE3) or TOP10 cells, where protein synthesis is possibly slower than proteolysis.
[0409] The data in all three expression systems (C43(DE3)/T7, BL21(DE3)/T7, TOP10/pBAD) demonstrate that fusions of HB to other common carrier proteins are highly susceptible to proteolysis. The non-cage forming HB-Enc fusions are also susceptible to proteolysis, but their expression is robust in all three expression systems, suggesting that 1) they are not as susceptible to proteolysis as the other fusions; and 2) the K71.sup.TEV containing Enc proteins may be providing some additional occlusion of the HB peptide to allow its expression. The cage-forming HB-Enc fusions fully protect the HB peptide from proteolysis, but their over-expression is not as robust because they appear to confer some toxicity to the expression strain upon over-expression.
[0410] Thus, there appears to be a trade-off between high over-expression and peptide release (non-cage forming HB-Enc) versus protection from proteolysis (cage forming HB-Enc).
Example 25
Enc Constructs Comprising M-Ap-
[0411] Ap was fused to the various engineered Enc examined in this study as well as typical carrier proteins SUMO, Trx, GST, and MBP and expression of the fusions from a T7 promoter in E. coli C43(DE3) cells was tested (FIG. 36). Fusions of Ap to the N-terminus of the various engineered Enc proteins were over-expressed in C43(DE3) cells, as well as Ap-SUMO, Ap-GST, and Ap-MBP. However, Ap-Trx and a C-terminal EncK138.sup.TEV-His-Ap fusion were not well expressed and could not be detected by SDS-PAGE. These data suggest that Enc works comparably to SUMO, GST, and MBP as a carrier protein for non-toxic Ap, but it must be fused to the N-terminus of Enc to enable Ap expression.
[0412] Interestingly, Ni-NTA purification of Ap-EncK71.sup.TEVK138.sup.TEV-His was achieved with high affinity binding of the protein to the resin (FIG. 37). In contrast, Ap-EncK138.sup.TEV-His, Ap-EncD60.sup.TEVK138.sup.TEV-His and Ap-EncV57.sup.TEVK138.sup.TEV-His did not bind well to the Ni-NTA resin and thus, needed to be purified using an alternative method of heat precipitation followed by ammonium sulfate precipitation. Characterization of the purified fusions by SEC, native PAGE, and TEM showed similar results to the HB-Enc fusions, where K71.sup.TEV containing Ap-Enc constructs did not form protein cages, whereas Ap-Enc without the K71.sup.TEV insertion were able to form cages (FIG. S8).
Example 26
Isolation of M-Ap-TEV Peptide Following TEV Protease Cleavage
[0413] In addition to HBCM2 peptide, engineered Enc constructs were provided including the proline-rich, unstructured AMP, apidaecin Ia (Ap) [64].
[0414] The Ap peptide was initially tested as a model antimicrobial peptide with intercellular toxic activity; proline-rich AMPS are generally bacteriostatic by inhibition of the ribosome [65]. However, we later discovered that Ap with a residual C-terminal cleaved TEV site (Ap-TEV) was found to lack bacteriostatic activity, unlike native Ap, suggesting that the residual TEV site interferes with Ap activity. We proceeded to test whether fusion of Enc to Ap helps its expression as a case study for a non-toxic peptide.
[0415] In particular the engineered Enc construct Ap-EncK71.sup.TEVK138.sup.TEV-His wherein 1) a TEV protease site followed by a GGT linker was placed between the C-terminus of Ap and the N-terminus of Enc; 2) a TEV protease site is inserted following residue K71 in Enc with GG-linkers on both the N- and C-termini of the insertion; and 3) a TEV protease site followed by a hexa-histidine tag is inserted following residue K138 in Enc with G.sub.5-linkers on both the N- and C-termini of the insertion.
[0416] The engineered Enc construct Ap-EncK71.sup.TEVK138.sup.TEV-His was tested for expression in C43(DE3) cells.
[0417] In particular, purified Ap-EncK71.sup.TEVK138.sup.TEV-His was digested with TEV protease as described in Example 14. Following overnight digestion at 4.degree. C., digested material was filtered through a centrifugal filter with 10 kDa molecular weight cutoff (Vivaspin, Satorius). Peptide was recovered in the filtrate. Material in the filter (<500 .mu.L) was diluted an additional two times to 5 mL using phosphate buffer (25 mM sodium phosphate pH 7.5, 100 mM NaCl) and centrifuged to collect additional filtrate. All filtrate was pooled and lyophilized. Final purified material was analyzed by SDS-PAGE and quantified by absorbance at 280 nm.
[0418] Ultimately, an overall yield of 3.5 mg/L culture of Ap-TEV peptide was obtained from 43 mg/L of Ap-EncK71.sup.TEVK138.sup.TEV-His fusion protein (FIG. 35). These data collectively show that Enc can aid expression and purification of a non-toxic peptide.
Example 27
Prophetic Example of Engineered Constructs Designed to Allow Post Isolation Digestion of the Residual Protease Cleavage Site Attached to the C-Terminus of the Target Proteins
[0419] In an example target protein (abbreviated TP) where a residual protease cleavage site on the C-terminus of TP interferes with the activity of TP, the following methods may be conducted to remove the majority of the residual site, leaving only a proline residue at the C-terminus of TP. Removal of residual site may possibly restore the activity/function of the TP.
[0420] First, a fused TP-Enc construct will need to be re-designed such that a proline residue is inserted between the C-terminus of TP and the N-terminus of the adjacent protease cleavage site. The new TP-Enc construct can then be over-expressed in C43(DE3) E. coli cells as described in Example 12, purified as described in Example 19, and digested with an appropriate protease as described in Example 14 and 19.
[0421] Following digestion, the released TP can be isolated by size exclusion chromatography or a centrifugal filter with an appropriate molecular weight cutoff. Alternatively, released TP can is isolated using ion exchange chromatography or reverse-phase (e.g., C18) chromatography, if appropriate.
[0422] Purified released TP can then be digested with commercially available carboxypeptidase A and/or B, according to manufacturer's instructions. Following carboxypeptidase digestion, TP with a residual proline at its C-terminus can be re-isolated using the same methods as in [00379].
Example 28
Isolation and Detection of Microcompartments from Bacteria
[0423] Cages from bacteria can be isolated by re-suspending cells in buffer and lysing the suspended cells. For example, cells can be re-suspended in a buffer such as 50 mM NaH.sub.2PO.sub.4, pH 8.0 with 200 mM NaCl and lysed by French pressure cell at 14,000 psi. Following removal of insoluble material by centrifugation at 12,000 g, 4.degree. C. for 15 min, the supernatant is heated at 70-85.degree. C. for 10 min. Only encapsulin cages will remain soluble under these conditions and insoluble material can again be removed by centrifugation. Ammonium sulfate precipitation at 25 or 50% can be performed to further purify the material, followed by size exclusion chromatography (SEC). SEC can be done using SHIMADZU FPLC with a Superose 6 increase 3.2/300 column (GE biosciences). One to 5 mg of sample is loaded, and protein is eluted with 50 mM NaH.sub.2PO.sub.4, pH 8.0, 200 mM NaCl at a flow rate of 0.2 mL/min for 20 min. Protein cages should elute as a high molecular weight species near the void volume, between 7 and 8 min.
[0424] Protein cages collected after SEC can be detected using transmission electron microscopy. For example, protein can be spotted on a copper TEM grid coated with carbon type B-formvar and stained using 2% uranyl nitrate, using standard methods. Grids can be examined on a transmission electron microscope (e.g., FEI Titan) at 80 kV. Hexagonal species of .about.25 nm diameter is indicative of cage formation.
[0425] Observation of (1) a high molecular species by SEC and (2) hexagonal cage features by TEM confirms cage formation.
[0426] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
[0427] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
[0428] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.
[0429] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.
[0430] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
[0431] When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
[0432] A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods may include a large number of optional composition and processing elements and steps.
[0433] In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
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Sequence CWU
1
1
1431252PRTArtificial SequenceSynthetic polypeptidemisc_feature(1)..(1)X1
is Mmisc_feature(2)..(2)X2 is Dmisc_feature(3)..(3)X3 is
Nmisc_feature(4)..(4)X4 is Lmisc_feature(5)..(5)X5 is
Kmisc_feature(6)..(6)X6 is Rmisc_feature(7)..(7)iX7 is
Emisc_feature(8)..(8)X8 is Lmisc_feature(9)..(9)X9 is
Amisc_feature(10)..(10)X10 is Pmisc_feature(11)..(11)X11 is
Lmisc_feature(12)..(12)X12 is Tmisc_feature(13)..(13)X13 is
Emisc_feature(14)..(14)X14 is Emisc_feature(15)..(15)X15 is
Amisc_feature(16)..(16)X16 is Wmisc_feature(17)..(17)X17 is
Amisc_feature(18)..(18)X18 is Emisc_feature(19)..(19)X19 is
Imisc_feature(20)..(20)X20 is Dmisc_feature(21)..(21)X21 is
Emisc_feature(22)..(22)X22 is Emisc_feature(23)..(23)X23 is
Amisc_feature(24)..(24)X24 is Rmisc_feature(25)..(25)X25 is
Emisc_feature(26)..(26)X26 is Tmisc_feature(27)..(27)X27 is
Amisc_feature(28)..(28)X28 is Kmisc_feature(29)..(29)X29 is
Rmisc_feature(30)..(30)X30 is Hmisc_feature(31)..(31)X31 is
Lmisc_feature(32)..(32)X32 is Amisc_feature(33)..(33)X33 is
Gmisc_feature(34)..(34)X34 is Rmisc_feature(35)..(35)X35 is
Rmisc_feature(36)..(36)X36 is Vmisc_feature(37)..(37)X37 is
Vmisc_feature(38)..(38)X38 is Dmisc_feature(39)..(39)X39 is
Vmisc_feature(40)..(40)X40 is Emisc_feature(41)..(41)X41 is
Gmisc_feature(42)..(42)X42 is Pmisc_feature(43)..(43)X43 is
Lmisc_feature(44)..(44)X44 is Gmisc_feature(45)..(45)X45 is
Wmisc_feature(46)..(46)X46 is Gmisc_feature(47)..(47)X47 is
Ymisc_feature(48)..(48)X48 is Smisc_feature(49)..(49)X49 is
Amisc_feature(50)..(50)X50 is Vmisc_feature(51)..(51)X51 is
Pmisc_feature(52)..(52)X52 is Lmisc_feature(53)..(53)X53 is
Gmisc_feature(54)..(54)X54 is Rmisc_feature(55)..(55)X55 is
Lmisc_feature(56)..(56)X56 is Emisc_feature(57)..(57)X57 is
Emisc_feature(58)..(58)X58 is Imisc_feature(59)..(59)X59 is
Emisc_feature(60)..(60)X60 is Gmisc_feature(61)..(61)X61 is
Pmisc_feature(62)..(62)X62 is Amisc_feature(63)..(63)X63 is
Emisc_feature(64)..(64)X64 is Gmisc_feature(65)..(65)X65 is
Vmisc_feature(66)..(66)X66 is Qmisc_feature(67)..(67)X67 is
Amisc_feature(68)..(68)X68 is Gmisc_feature(69)..(69)X69 is
Vmisc_feature(70)..(70)X70 is Rmisc_feature(71)..(71)X71 is
Qmisc_feature(72)..(72)X72 is Vmisc_feature(73)..(73)X73 is
Lmisc_feature(74)..(74)X74 is Pmisc_feature(75)..(75)X75 is
Lmisc_feature(76)..(76)X76 is Pmisc_feature(77)..(77)X77 is
Emisc_feature(78)..(78)X78 is Lmisc_feature(79)..(79)X79 is
Rmisc_feature(80)..(80)X80 is Vmisc_feature(81)..(81)X81 is
Pmisc_feature(82)..(82)X82 is Fmisc_feature(83)..(83)X83 is
Tmisc_feature(84)..(84)X84 is Lmisc_feature(85)..(85)X85 is
Smisc_feature(86)..(86)X86 is Rmisc_feature(87)..(87)X87 is
Rmisc_feature(88)..(88)X88 is Dmisc_feature(89)..(89)X89 is
Lmisc_feature(90)..(90)X90 is Dmisc_feature(91)..(91)X91 is
Amisc_feature(92)..(92)X92 is Vmisc_feature(93)..(93)X93 is
Emisc_feature(94)..(94)X94 is Rmisc_feature(95)..(95)X95 is
Gmisc_feature(96)..(96)X96 is Amisc_feature(97)..(97)X97 is
Kmisc_feature(98)..(98)X98 is Dmisc_feature(99)..(99)X99 is
Lmisc_feature(100)..(100)X100 is Dmisc_feature(101)..(101)X101 is
Lmisc_feature(102)..(102)X102 is Smisc_feature(103)..(103)X103 is
Pmisc_feature(104)..(104)X104 is Vmisc_feature(105)..(105)X105 is
Amisc_feature(106)..(106)X106 is Emisc_feature(107)..(107)X107 is
Amisc_feature(108)..(108)X108 is Amisc_feature(109)..(109)X109 is
Rmisc_feature(110)..(110)X110 is Lmisc_feature(111)..(111)X111 is
Lmisc_feature(112)..(112)X112 is Amisc_feature(113)..(113)X113 is
Rmisc_feature(114)..(114)X114 is Amisc_feature(115)..(115)X115 is
Emisc_feature(116)..(116)X116 is Dmisc_feature(117)..(117)X117 is
Rmisc_feature(118)..(118)X118 is Lmisc_feature(119)..(119)X119 is
Imisc_feature(120)..(120)X120 is Fmisc_feature(121)..(121)X121 is
Nmisc_feature(122)..(122)X122 is Gmisc_feature(123)..(123)X123 is
Ymisc_feature(124)..(124)X124 is Amisc_feature(125)..(125)X125 is
Emisc_feature(126)..(126)X126 is Amisc_feature(127)..(127)X127 is
Gmisc_feature(128)..(128)X128 is Imisc_feature(129)..(129)X129 is
Emisc_feature(130)..(130)X130 is Gmisc_feature(131)..(131)X131 is
Lmisc_feature(132)..(132)X132 is Lmisc_feature(133)..(133)X133 is
Nmisc_feature(134)..(134)X134 is Amisc_feature(135)..(135)X135 is
Smisc_feature(136)..(136)X136 is Gmisc_feature(137)..(137)X137 is
Nmisc_feature(138)..(138)X138 is Lmisc_feature(139)..(139)X139 is
Kmisc_feature(140)..(140)X140 is Lmisc_feature(141)..(141)X141 is
Pmisc_feature(142)..(142)X142 is Lmisc_feature(143)..(143)X143 is
Smisc_feature(144)..(144)X144 is Amisc_feature(145)..(145)X145 is
Dmisc_feature(146)..(146)X146 is Pmisc_feature(147)..(147)X147 is
Gmisc_feature(148)..(148)X148 is Dmisc_feature(149)..(149)X149 is
Imisc_feature(150)..(150)X150 is Pmisc_feature(151)..(151)X151 is
Dmisc_feature(152)..(152)X152 is Amisc_feature(153)..(153)X153 is
Imisc_feature(154)..(154)X154 is Amisc_feature(155)..(155)X155 is
Emisc_feature(156)..(156)X156 is Amisc_feature(157)..(157)X157 is
Lmisc_feature(158)..(158)X158 is Tmisc_feature(159)..(159)X159 is
Kmisc_feature(160)..(160)X160 is Lmisc_feature(161)..(161)X161 is
Rmisc_feature(162)..(162)X162 is Emisc_feature(163)..(163)X163 is
Amisc_feature(164)..(164)X164 is Gmisc_feature(165)..(165)X165 is
Vmisc_feature(166)..(166)X166 is Emisc_feature(167)..(167)X167 is
Gmisc_feature(168)..(168)X168 is Pmisc_feature(169)..(169)X169 is
Ymisc_feature(170)..(170)X170 is Amisc_feature(171)..(171)X171 is
Lmisc_feature(172)..(172)X172 is Vmisc_feature(173)..(173)X173 is
Lmisc_feature(174)..(174)X174 is Smisc_feature(175)..(175)X175 is
Pmisc_feature(176)..(176)X176 is Dmisc_feature(177)..(177)X177 is
Lmisc_feature(178)..(178)X178 is Ymisc_feature(179)..(179)X179 is
Tmisc_feature(180)..(180)X180 is Amisc_feature(181)..(181)X181 is
Lmisc_feature(182)..(182)X182 is Fmisc_feature(183)..(183)X183 is
Rmisc_feature(184)..(184)X184 is Vmisc_feature(185)..(185)X185 is
Ymisc_feature(186)..(186)X186 is Dmisc_feature(187)..(187)X187 is
Gmisc_feature(188)..(188)X188 is Tmisc_feature(189)..(189)X189 is
Gmisc_feature(190)..(190)X190 is Ymisc_feature(191)..(191)X191 is
Pmisc_feature(192)..(192)X192 is Emisc_feature(193)..(193)X193 is
Imisc_feature(194)..(194)X194 is Emisc_feature(195)..(195)X195 is
Hmisc_feature(196)..(196)X196 is Imisc_feature(197)..(197)X197 is
Kmisc_feature(198)..(198)X198 is Emisc_feature(199)..(199)X199 is
Lmisc_feature(200)..(200)X200 is Vmisc_feature(201)..(201)X201 is
Dmisc_feature(202)..(202)X202 is Gmisc_feature(203)..(203)X203 is
Gmisc_feature(204)..(204)X204 is Vmisc_feature(205)..(205)X205 is
Imisc_feature(206)..(206)X206 is Wmisc_feature(207)..(207)X207 is
Amisc_feature(208)..(208)X208 is Pmisc_feature(209)..(209)X209 is
Amisc_feature(210)..(210)X210 is Lmisc_feature(211)..(211)X211 is
Dmisc_feature(212)..(212)X212 is Gmisc_feature(213)..(213)X213 is
Gmisc_feature(214)..(214)X214 is Amisc_feature(215)..(215)X215 is
Vmisc_feature(216)..(216)X216 is Lmisc_feature(217)..(217)X217 is
Vmisc_feature(218)..(218)X218 is Smisc_feature(219)..(219)X219 is
Tmisc_feature(220)..(220)X220 is Rmisc_feature(221)..(221)X221 is
Gmisc_feature(222)..(222)X222 is Gmisc_feature(223)..(223)X223 is
Dmisc_feature(224)..(224)X224 is Fmisc_feature(225)..(225)X225 is
Dmisc_feature(226)..(226)X226 is Lmisc_feature(227)..(227)X227 is
Tmisc_feature(228)..(228)X228 is Lmisc_feature(229)..(229)X229 is
Gmisc_feature(230)..(230)X230 is Qmisc_feature(231)..(231)X231 is
Dmisc_feature(232)..(232)X232 is Lmisc_feature(233)..(233)X233 is
Smisc_feature(234)..(234)X234 is Imisc_feature(235)..(235)X235 is
Gmisc_feature(236)..(236)X236 is Ymisc_feature(237)..(237)X237 is
Lmisc_feature(238)..(238)X238 is Smisc_feature(239)..(239)X239 is
Hmisc_feature(240)..(240)X240 is Dmisc_feature(241)..(241)X241 is
Amisc_feature(242)..(242)X242 is Dmisc_feature(243)..(243)X243 is
Nmisc_feature(244)..(244)X244 is Vmisc_feature(245)..(245)X245 is
Emisc_feature(246)..(246)X246 is Lmisc_feature(247)..(247)X247 is
Fmisc_feature(248)..(248)X248 is Lmisc_feature(249)..(249)X249 is
Tmisc_feature(250)..(250)Xaa can be any naturally occurring amino
acidmisc_feature(251)..(251)X251 is Smisc_feature(252)..(252)X252 is
Fmisc_feature(253)..(253)X250 is Emisc_feature(253)..(253)X253 is T 1Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa65 70 75
80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100
105 110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130
135 140Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa145 150 155
160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
165 170 175Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180
185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 195 200 205Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210
215 220Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa225 230 235
240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
245 2502253PRTArtificial SequenceSynthetic polypeptide
2Met Asp Asn Leu Lys Arg Glu Leu Ala Pro Leu Thr Glu Glu Ala Trp1
5 10 15Ala Glu Ile Asp Glu Glu
Ala Arg Glu Thr Ala Lys Arg His Leu Ala 20 25
30Gly Arg Arg Val Val Asp Val Glu Gly Pro Leu Gly Trp
Gly Tyr Ser 35 40 45Ala Val Pro
Leu Gly Arg Leu Glu Glu Ile Glu Gly Pro Ala Glu Gly 50
55 60Val Gln Ala Gly Val Arg Gln Val Leu Pro Leu Pro
Glu Leu Arg Val65 70 75
80Pro Phe Thr Leu Ser Arg Arg Asp Leu Asp Ala Val Glu Arg Gly Ala
85 90 95Lys Asp Leu Asp Leu Ser
Pro Val Ala Glu Ala Ala Arg Lys Leu Ala 100
105 110Arg Ala Glu Asp Arg Leu Ile Phe Asn Gly Tyr Ala
Glu Ala Gly Ile 115 120 125Glu Gly
Leu Leu Asn Ala Ser Gly Asn Leu Lys Leu Pro Leu Ser Ala 130
135 140Asp Pro Gly Asp Ile Pro Asp Ala Ile Ala Glu
Ala Leu Thr Lys Leu145 150 155
160Arg Glu Ala Gly Val Glu Gly Pro Tyr Ala Leu Val Leu Ser Pro Asp
165 170 175Leu Tyr Thr Ala
Leu Phe Arg Val Tyr Asp Gly Thr Gly Tyr Pro Glu 180
185 190Ile Glu His Ile Lys Glu Leu Val Asp Gly Gly
Val Ile Trp Ala Pro 195 200 205Ala
Leu Asp Gly Gly Ala Val Leu Val Ser Thr Arg Gly Gly Asp Phe 210
215 220Asp Leu Thr Leu Gly Gln Asp Leu Ser Ile
Gly Tyr Leu Ser His Asp225 230 235
240Ala Asp Asn Val Glu Leu Phe Leu Thr Glu Ser Phe Thr
245 250345PRTArtificial SequenceSynthetic
polypeptide 3Asp Asn Leu Lys Arg Glu Leu Ala Pro Leu Thr Glu Glu Ala Trp
Ala1 5 10 15Glu Ile Asp
Glu Glu Ala Arg Glu Thr Ala Lys Arg His Leu Ala Gly 20
25 30Arg Arg Val Val Asp Val Glu Gly Pro Leu
Gly Trp Gly 35 40
45428PRTArtificial SequenceSynthetic polypeptide 4Tyr Ser Ala Val Pro Leu
Gly Arg Leu Glu Glu Ile Glu Gly Pro Ala1 5
10 15Glu Gly Val Gln Ala Gly Val Arg Gln Val Leu Pro
20 25556PRTArtificial SequenceSynthetic
polypeptide 5Leu Pro Glu Leu Arg Val Pro Phe Thr Leu Ser Arg Arg Asp Leu
Asp1 5 10 15Ala Val Glu
Arg Gly Ala Lys Asp Leu Asp Leu Ser Pro Val Ala Glu 20
25 30Ala Ala Arg Lys Leu Ala Arg Ala Glu Asp
Arg Leu Ile Phe Asn Gly 35 40
45Tyr Ala Glu Ala Gly Ile Glu Gly 50
55690PRTArtificial SequenceSynthetic polypeptide 6Leu Leu Asn Ala Ser Gly
Asn Leu Lys Leu Pro Leu Ser Ala Asp Pro1 5
10 15Gly Asp Ile Pro Asp Ala Ile Ala Glu Ala Leu Thr
Lys Leu Arg Glu 20 25 30Ala
Gly Val Glu Gly Pro Tyr Ala Leu Val Leu Ser Pro Asp Leu Tyr 35
40 45Thr Ala Leu Phe Arg Val Tyr Asp Gly
Thr Gly Tyr Pro Glu Ile Glu 50 55
60His Ile Lys Glu Leu Val Asp Gly Gly Val Ile Trp Ala Pro Ala Leu65
70 75 80Asp Gly Gly Ala Val
Leu Val Ser Thr Arg 85 90733PRTT.
maritima 7Gly Gly Asp Phe Asp Leu Thr Leu Gly Gln Asp Leu Ser Ile Gly
Tyr1 5 10 15Leu Ser His
Asp Ala Asp Asn Val Glu Leu Phe Leu Thr Glu Ser Phe 20
25 30Thr85PRTArtificial SequenceSynthetic
polypeptidemisc_featurewherein X1=D or Nmisc_featureX can be any amino
acidmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino acid
8Ala Xaa Leu Xaa Xaa1 5926PRTArtificial SequenceSynthetic
polypeptide 9Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys
Val1 5 10 15Val Thr Thr
Ala Lys Lys Pro Leu Ile Val 20
251027PRTArtificial SequenceSynthetic polypeptide 10Lys Trp Lys Lys Phe
Ile Lys Ser Leu Thr Lys Ser Ala Ala Lys Thr1 5
10 15Val Val Lys Thr Ala Lys Lys Pro Leu Ile Val
20 251139PRTArtificial SequenceSynthetic
polypeptide 11Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro
Pro1 5 10 15Phe Phe Pro
Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20
25 30Arg Phe Pro Pro Arg Phe Pro
351218PRTArtificial SequenceSynthetic polypeptide 12Gly Asn Asn Arg Pro
Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro1 5
10 15Arg Ile1310PRTArtificial SequenceSynthetic
polypeptide 13Leu Glu Val Leu Phe Gln Gly Pro Gly Pro1 5
10145PRTArtificial SequenceSynthetic polypeptide 14Asp Asp
Asp Asp Lys1 5154PRTArtificial SequenceSynthetic
polypeptide 15Ile Glu Gly Arg1167PRTArtificial SequenceSynthetic
polypeptide 16Glu Asn Leu Tyr Phe Gln Gly1
5176PRTArtificial SequenceSynthetic polypeptide 17Leu Val Pro Arg Gly
Ser1 518320PRTArtificial SequenceSynthetic polypeptide
18Met Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His1
5 10 15Pro Arg Ile Glu Asn Leu
Tyr Phe Gln Gly Gly Thr Ser Glu Phe Leu 20 25
30Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp Gln
Glu Ile Asp 35 40 45Asn Arg Ala
Arg Glu Ile Phe Lys Thr Gln Leu Tyr Gly Arg Lys Phe 50
55 60Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala
Ala His Pro Leu65 70 75
80Gly Glu Val Glu Val Leu Ser Asp Glu Asn Glu Val Val Lys Trp Gly
85 90 95Leu Arg Lys Ser Leu Pro
Leu Ile Glu Leu Arg Ala Thr Phe Thr Leu 100
105 110Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys
Pro Asn Val Asp 115 120 125Leu Ser
Ser Leu Glu Glu Thr Val Arg Lys Val Ala Glu Phe Glu Asp 130
135 140Glu Val Ile Phe Arg Gly Cys Glu Lys Ser Gly
Val Lys Gly Leu Leu145 150 155
160Ser Phe Glu Glu Arg Lys Gly Gly Gly Gly Gly Glu Asn Leu Tyr Phe
165 170 175Gln Gly His His
His His His His Gly Gly Gly Gly Gly Ile Glu Cys 180
185 190Gly Ser Thr Pro Lys Asp Leu Leu Glu Ala Ile
Val Arg Ala Leu Ser 195 200 205Ile
Phe Ser Lys Asp Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn 210
215 220Thr Asp Arg Trp Ile Asn Phe Leu Lys Glu
Glu Ala Gly His Tyr Pro225 230 235
240Leu Glu Lys Arg Val Glu Glu Cys Leu Arg Gly Gly Lys Ile Ile
Thr 245 250 255Thr Pro Arg
Ile Glu Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp 260
265 270Phe Lys Leu Ile Leu Gly Gln Asp Leu Ser
Ile Gly Tyr Glu Asp Arg 275 280
285Glu Lys Asp Ala Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln 290
295 300Val Val Asn Pro Glu Ala Leu Ile
Leu Leu Lys Phe Ser Gly Gly Ser305 310
315 3201924PRTArtificial SequenceSynthetic polypeptide
19Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro1
5 10 15Arg Ile Glu Asn Leu Tyr
Phe Gln 202037PRTArtificial SequenceSynthetic polypeptide
20Lys Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly Gln Asn Ile Arg1
5 10 15Asp Gly Ile Ile Lys Ala
Gly Pro Ala Val Ala Val Val Gly Gln Ala 20 25
30Thr Gln Ile Ala Lys 352135PRTArtificial
SequenceSynthetic polypeptide 21Lys Trp Lys Val Phe Lys Lys Ile Glu Lys
Met Gly Arg Asn Ile Arg1 5 10
15Asn Gly Ile Val Lys Ala Gly Pro Ala Ile Ala Val Leu Gly Glu Ala
20 25 30Lys Ala Leu
352226PRTArtificial SequenceSynthetic polypeptide 22Gly Ile Gly Ala Val
Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu1 5
10 15Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 2523315PRTArtificial SequenceSynthetic
polypeptide 23Met Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro
His1 5 10 15Pro Arg Ile
Glu Asn Leu Tyr Phe Gln Gly Gly Thr Ser Pro Asp Phe 20
25 30Leu Gly His Ala Glu Asn Pro Leu Arg Glu
Glu Glu Trp Ala Arg Leu 35 40
45Asn Glu Thr Val Ile Gln Val Ala Arg Arg Ser Leu Val Gly Arg Arg 50
55 60Ile Leu Asp Ile Tyr Gly Pro Leu Gly
Ala Gly Val Gln Thr Val Pro65 70 75
80Tyr Asp Glu Phe Gln Gly Val Ser Pro Gly Ala Val Asp Ile
Val Gly 85 90 95Glu Gln
Glu Thr Ala Met Val Phe Thr Asp Ala Arg Lys Phe Lys Thr 100
105 110Ile Pro Ile Ile Tyr Lys Asp Phe Leu
Leu His Trp Arg Asp Ile Glu 115 120
125Ala Ala Arg Thr His Asn Met Pro Leu Asp Val Ser Ala Ala Ala Gly
130 135 140Ala Ala Ala Leu Cys Ala Gln
Gln Glu Asp Glu Leu Ile Phe Tyr Gly145 150
155 160Asp Ala Arg Leu Gly Tyr Glu Gly Leu Met Thr Ala
Asn Gly Arg Leu 165 170
175Thr Val Pro Leu Gly Asp Trp Thr Ser Pro Gly Gly Gly Phe Gln Ala
180 185 190Ile Val Glu Ala Thr Arg
Lys Leu Asn Glu Gln Gly His Phe Gly Pro 195 200
205Tyr Ala Val Val Leu Ser Pro Arg Leu Tyr Ser Gln Leu His
Arg Ile 210 215 220Tyr Glu Lys Thr Gly
Val Leu Glu Ile Glu Thr Ile Arg Gln Leu Ala225 230
235 240Ser Asp Gly Val Tyr Gln Ser Asn Arg Leu
Arg Gly Glu Ser Gly Val 245 250
255Val Val Ser Thr Gly Arg Glu Asn Met Asp Leu Ala Val Ser Met Asp
260 265 270Met Val Ala Ala Tyr
Leu Gly Ala Ser Arg Met Asn His Pro Phe Arg 275
280 285Val Leu Glu Ala Leu Leu Leu Arg Ile Lys His Pro
Asp Ala Ile Cys 290 295 300Thr Leu Glu
Gly Ala Gly Ala Thr Glu Arg Arg305 310
3152415PRTArtificial SequenceSynthetic polypeptide 24Gly Leu Asn Asp Ile
Phe Glu Ala Gln Lys Ile Glu Trp His Glu1 5
10 152526PRTArtificial SequenceSynthetic polypeptide
25Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg1
5 10 15Phe Lys Lys Ile Ser Ser
Ser Gly Ala Leu 20 25266PRTArtificial
SequenceSynthetic polypeptide 26Glu Glu Glu Glu Glu Glu1
52713PRTArtificial SequenceSynthetic polypeptide 27Gly Ala Pro Val Pro
Tyr Pro Asp Pro Leu Glu Pro Arg1 5
10288PRTArtificial SequenceSynthetic polypeptide 28Asp Tyr Lys Asp Asp
Asp Asp Lys1 5299PRTArtificial SequenceSynthetic
polypeptide 29Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1
5306PRTArtificial SequenceSynthetic polypeptide 30His His His His His
His1 53110PRTArtificial SequenceSynthetic polypeptide 31Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu1 5
103218PRTArtificial SequenceSynthetic polypeptide 32Thr Lys Glu Asn Pro
Arg Ser Asn Gln Glu Glu Ser Tyr Asp Asp Asn1 5
10 15Glu Ser3315PRTArtificial SequenceSynthetic
polypeptide 33Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
Ser1 5 10
153438PRTArtificial SequenceSynthetic polypeptide 34Met Asp Glu Lys Thr
Thr Gly Trp Arg Gly Gly His Val Val Glu Gly1 5
10 15Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg
Leu Glu His His Pro 20 25
30Gln Gly Gln Arg Glu Pro 353513PRTArtificial SequenceSynthetic
polypeptide 35Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser1
5 10368PRTArtificial SequenceSynthetic
polypeptide 36Thr Gln Asp Pro Ser Arg Val Gly1
5378PRTArtificial SequenceSynthetic polypeptide 37Trp Ser His Pro Gln Phe
Glu Lys1 5386PRTArtificial SequenceSynthetic polypeptide
38Cys Cys Pro Gly Cys Cys1 53914PRTArtificial
SequenceSynthetic polypeptide 39Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly
Leu Asp Ser Thr1 5 104011PRTArtificial
SequenceSynthetic polypeptide 40Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly
Lys1 5 10418PRTArtificial
SequenceSynthetic polypeptide 41Asp Leu Tyr Asp Asp Asp Asp Lys1
54216PRTArtificial SequenceSynthetic polypeptide 42Thr Asp Lys Asp
Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu1 5
10 154313PRTArtificial SequenceSynthetic
polypeptide 43Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys1
5 104412PRTArtificial SequenceSynthetic
polypeptide 44Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys1
5 1045264PRTT. maritima 45Glu Phe Leu Lys Arg Ser
Phe Ala Pro Leu Thr Glu Lys Gln Trp Gln1 5
10 15Glu Ile Asp Asn Arg Ala Arg Glu Ile Phe Lys Thr
Gln Leu Tyr Gly 20 25 30Arg
Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala Ala 35
40 45His Pro Leu Gly Glu Val Glu Val Leu
Ser Asp Glu Asn Glu Val Val 50 55
60Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu Arg Ala Thr65
70 75 80Phe Thr Leu Asp Leu
Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys Pro 85
90 95Asn Val Asp Leu Ser Ser Leu Glu Glu Thr Val
Arg Lys Val Ala Glu 100 105
110Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser Gly Val Lys
115 120 125Gly Leu Leu Ser Phe Glu Glu
Arg Lys Ile Glu Cys Gly Ser Thr Pro 130 135
140Lys Asp Leu Leu Glu Ala Ile Val Arg Ala Leu Ser Ile Phe Ser
Lys145 150 155 160Asp Gly
Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr Asp Arg Trp
165 170 175Ile Asn Phe Leu Lys Glu Glu
Ala Gly His Tyr Pro Leu Glu Lys Arg 180 185
190Val Glu Glu Cys Leu Arg Gly Gly Lys Ile Ile Thr Thr Pro
Arg Ile 195 200 205Glu Asp Ala Leu
Val Val Ser Glu Arg Gly Gly Asp Phe Lys Leu Ile 210
215 220Leu Gly Gln Asp Leu Ser Ile Gly Tyr Glu Asp Arg
Glu Lys Asp Ala225 230 235
240Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln Val Val Asn Pro
245 250 255Glu Ala Leu Ile Leu
Leu Lys Phe 26046277PRTM. xanthus 46Asp Phe Leu Gly His Ala
Glu Asn Pro Leu Arg Glu Glu Glu Trp Ala1 5
10 15Arg Leu Asn Glu Thr Val Ile Gln Val Ala Arg Arg
Ser Leu Val Gly 20 25 30Arg
Arg Ile Leu Asp Ile Tyr Gly Pro Leu Gly Ala Gly Val Gln Thr 35
40 45Val Pro Tyr Asp Glu Phe Gln Gly Val
Ser Pro Gly Ala Val Asp Ile 50 55
60Val Gly Glu Gln Glu Thr Ala Met Val Phe Thr Asp Ala Arg Lys Phe65
70 75 80Lys Thr Ile Pro Ile
Ile Tyr Lys Asp Phe Leu Leu His Trp Arg Asp 85
90 95Ile Glu Ala Ala Arg Thr His Asn Met Pro Leu
Asp Val Ser Ala Ala 100 105
110Ala Gly Ala Ala Ala Leu Cys Ala Gln Gln Glu Asp Glu Leu Ile Phe
115 120 125Tyr Gly Asp Ala Arg Leu Gly
Tyr Glu Gly Leu Met Thr Ala Asn Gly 130 135
140Arg Leu Thr Val Pro Leu Gly Asp Trp Thr Ser Pro Gly Gly Gly
Phe145 150 155 160Gln Ala
Ile Val Glu Ala Thr Arg Lys Leu Asn Glu Gln Gly His Phe
165 170 175Gly Pro Tyr Ala Val Val Leu
Ser Pro Arg Leu Tyr Ser Gln Leu His 180 185
190Arg Ile Tyr Glu Lys Thr Gly Val Leu Glu Ile Glu Thr Ile
Arg Gln 195 200 205Leu Ala Ser Asp
Gly Val Tyr Gln Ser Asn Arg Leu Arg Gly Glu Ser 210
215 220Gly Val Val Val Ser Thr Gly Arg Glu Asn Met Asp
Leu Ala Val Ser225 230 235
240Met Asp Met Val Ala Ala Tyr Leu Gly Ala Ser Arg Met Asn His Pro
245 250 255Phe Arg Val Leu Glu
Ala Leu Leu Leu Arg Ile Lys His Pro Asp Ala 260
265 270Ile Cys Thr Leu Glu 27547269PRTT. maritima
47Met Glu Phe Leu Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp1
5 10 15Gln Glu Ile Asp Asn Arg
Ala Arg Glu Ile Phe Lys Thr Gln Leu Tyr 20 25
30Gly Arg Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp
Glu Tyr Ala 35 40 45Ala His Pro
Leu Gly Glu Val Glu Val Leu Ser Asp Glu Asn Glu Val 50
55 60Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile
Glu Leu Arg Ala65 70 75
80Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys
85 90 95Pro Asn Val Asp Leu Ser
Ser Leu Glu Glu Thr Val Arg Lys Val Ala 100
105 110Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu
Lys Ser Gly Val 115 120 125Lys Gly
Leu Leu Ser Phe Glu Glu Arg Lys Ile Glu Cys Gly Ser Thr 130
135 140Pro Lys Asp Leu Leu Glu Ala Ile Val Arg Ala
Leu Ser Ile Phe Ser145 150 155
160Lys Asp Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr Asp Arg
165 170 175Trp Ile Asn Phe
Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys 180
185 190Arg Val Glu Glu Cys Leu Arg Gly Gly Lys Ile
Ile Thr Thr Pro Arg 195 200 205Ile
Glu Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe Lys Leu 210
215 220Ile Leu Gly Gln Asp Leu Ser Ile Gly Tyr
Glu Asp Arg Glu Lys Asp225 230 235
240Ala Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln Val Val
Asn 245 250 255Pro Glu Ala
Leu Ile Leu Leu Lys Phe Ser Gly Gly Ser 260
26548286PRTArtificial SequenceSynthetic polypeptide 48Met Ser Glu Phe Leu
Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln1 5
10 15Trp Gln Glu Ile Asp Asn Arg Ala Arg Glu Ile
Phe Lys Thr Gln Leu 20 25
30Tyr Gly Arg Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr
35 40 45Ala Ala His Pro Leu Gly Glu Val
Glu Val Leu Ser Asp Glu Asn Glu 50 55
60Val Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu Arg65
70 75 80Ala Thr Phe Thr Leu
Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly 85
90 95Lys Pro Asn Val Asp Leu Ser Ser Leu Glu Glu
Thr Val Arg Lys Val 100 105
110Ala Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser Gly
115 120 125Val Lys Gly Leu Leu Ser Phe
Glu Glu Arg Lys Gly Gly Gly Gly Gly 130 135
140His His His His His His Gly Gly Gly Gly Gly Ile Glu Cys Gly
Ser145 150 155 160Thr Pro
Lys Asp Leu Leu Glu Ala Ile Val Arg Ala Leu Ser Ile Phe
165 170 175Ser Lys Asp Gly Ile Glu Gly
Pro Tyr Thr Leu Val Ile Asn Thr Asp 180 185
190Arg Trp Ile Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro
Leu Glu 195 200 205Lys Arg Val Glu
Glu Cys Leu Arg Gly Gly Lys Ile Ile Thr Thr Pro 210
215 220Arg Ile Glu Asp Ala Leu Val Val Ser Glu Arg Gly
Gly Asp Phe Lys225 230 235
240Leu Ile Leu Gly Gln Asp Leu Ser Ile Gly Tyr Glu Asp Arg Glu Lys
245 250 255Asp Ala Val Arg Leu
Phe Ile Thr Glu Thr Phe Thr Phe Gln Val Val 260
265 270Asn Pro Glu Ala Leu Ile Leu Leu Lys Phe Ser Gly
Gly Ser 275 280
28549331PRTArtificial SequenceSynthetic polypeptide 49Met Lys Trp Lys Ser
Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1 5
10 15Val Val Thr Thr Ala Lys Lys Pro Leu Ile Val
Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Gly Gly Gly Thr Ser Glu Phe Leu Lys Arg Ser Phe Ala
35 40 45Pro Leu Thr Glu Lys Gln Trp Gln
Glu Ile Asp Asn Arg Ala Arg Glu 50 55
60Ile Phe Lys Thr Gln Leu Tyr Gly Arg Lys Phe Val Asp Val Glu Gly65
70 75 80Pro Tyr Gly Trp Glu
Tyr Ala Ala His Pro Leu Gly Glu Val Glu Val 85
90 95Leu Ser Asp Glu Asn Glu Val Val Lys Trp Gly
Leu Arg Lys Ser Leu 100 105
110Pro Leu Ile Glu Leu Arg Ala Thr Phe Thr Leu Asp Leu Trp Glu Leu
115 120 125Asp Asn Leu Glu Arg Gly Lys
Pro Asn Val Asp Leu Ser Ser Leu Glu 130 135
140Glu Thr Val Arg Lys Val Ala Glu Phe Glu Asp Glu Val Ile Phe
Arg145 150 155 160Gly Cys
Glu Lys Ser Gly Val Lys Gly Leu Leu Ser Phe Glu Glu Arg
165 170 175Lys Gly Gly Gly Gly Gly Glu
Asn Leu Tyr Phe Gln Gly His His His 180 185
190His His His Gly Gly Gly Gly Gly Ile Glu Cys Gly Ser Thr
Pro Lys 195 200 205Asp Leu Leu Glu
Ala Ile Val Arg Ala Leu Ser Ile Phe Ser Lys Asp 210
215 220Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr
Asp Arg Trp Ile225 230 235
240Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys Arg Val
245 250 255Glu Glu Cys Leu Arg
Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu 260
265 270Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe
Lys Leu Ile Leu 275 280 285Gly Gln
Asp Leu Ser Ile Gly Tyr Glu Asp Arg Glu Lys Asp Ala Val 290
295 300Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln
Val Val Asn Pro Glu305 310 315
320Ala Leu Ile Leu Leu Lys Phe Ser Gly Gly Ser 325
33050342PRTArtificial SequenceSynthetic polypeptide 50Met
Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1
5 10 15Val Val Thr Thr Ala Lys Lys
Pro Leu Ile Val Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Gly Gly Gly Thr Ser Glu Phe Leu Lys Arg Ser
Phe Ala 35 40 45Pro Leu Thr Glu
Lys Gln Trp Gln Glu Ile Asp Asn Arg Ala Arg Glu 50 55
60Ile Phe Lys Thr Gln Leu Tyr Gly Arg Lys Phe Val Asp
Val Glu Gly65 70 75
80Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro Leu Gly Glu Val Glu Val
85 90 95Leu Ser Asp Glu Asn Glu
Val Val Lys Trp Gly Leu Arg Lys Gly Gly 100
105 110Glu Asn Leu Tyr Phe Gln Gly Gly Gly Ser Leu Pro
Leu Ile Glu Leu 115 120 125Arg Ala
Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg 130
135 140Gly Lys Pro Asn Val Asp Leu Ser Ser Leu Glu
Glu Thr Val Arg Lys145 150 155
160Val Ala Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser
165 170 175Gly Val Lys Gly
Leu Leu Ser Phe Glu Glu Arg Lys Gly Gly Gly Gly 180
185 190Gly Glu Asn Leu Tyr Phe Gln Gly His His His
His His His Gly Gly 195 200 205Gly
Gly Gly Ile Glu Cys Gly Ser Thr Pro Lys Asp Leu Leu Glu Ala 210
215 220Ile Val Arg Ala Leu Ser Ile Phe Ser Lys
Asp Gly Ile Glu Gly Pro225 230 235
240Tyr Thr Leu Val Ile Asn Thr Asp Arg Trp Ile Asn Phe Leu Lys
Glu 245 250 255Glu Ala Gly
His Tyr Pro Leu Glu Lys Arg Val Glu Glu Cys Leu Arg 260
265 270Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile
Glu Asp Ala Leu Val Val 275 280
285Ser Glu Arg Gly Gly Asp Phe Lys Leu Ile Leu Gly Gln Asp Leu Ser 290
295 300Ile Gly Tyr Glu Asp Arg Glu Lys
Asp Ala Val Arg Leu Phe Ile Thr305 310
315 320Glu Thr Phe Thr Phe Gln Val Val Asn Pro Glu Ala
Leu Ile Leu Leu 325 330
335Lys Phe Ser Gly Gly Ser 34051342PRTArtificial
SequenceSynthetic polypeptide 51Met Lys Trp Lys Ser Phe Ile Lys Lys Leu
Thr Lys Ala Ala Lys Lys1 5 10
15Val Val Thr Thr Ala Lys Lys Pro Leu Ile Val Glu Asn Leu Tyr Phe
20 25 30Gln Gly Gly Gly Gly Gly
Thr Ser Glu Phe Leu Lys Arg Ser Phe Ala 35 40
45Pro Leu Thr Glu Lys Gln Trp Gln Glu Ile Asp Asn Arg Ala
Arg Glu 50 55 60Ile Phe Lys Thr Gln
Leu Tyr Gly Arg Lys Phe Val Asp Val Glu Gly65 70
75 80Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro
Leu Gly Glu Val Glu Val 85 90
95Leu Ser Asp Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Gly Glu Asn
100 105 110Glu Val Val Lys Trp
Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu 115
120 125Arg Ala Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp
Asn Leu Glu Arg 130 135 140Gly Lys Pro
Asn Val Asp Leu Ser Ser Leu Glu Glu Thr Val Arg Lys145
150 155 160Val Ala Glu Phe Glu Asp Glu
Val Ile Phe Arg Gly Cys Glu Lys Ser 165
170 175Gly Val Lys Gly Leu Leu Ser Phe Glu Glu Arg Lys
Gly Gly Gly Gly 180 185 190Gly
Glu Asn Leu Tyr Phe Gln Gly His His His His His His Gly Gly 195
200 205Gly Gly Gly Ile Glu Cys Gly Ser Thr
Pro Lys Asp Leu Leu Glu Ala 210 215
220Ile Val Arg Ala Leu Ser Ile Phe Ser Lys Asp Gly Ile Glu Gly Pro225
230 235 240Tyr Thr Leu Val
Ile Asn Thr Asp Arg Trp Ile Asn Phe Leu Lys Glu 245
250 255Glu Ala Gly His Tyr Pro Leu Glu Lys Arg
Val Glu Glu Cys Leu Arg 260 265
270Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu Asp Ala Leu Val Val
275 280 285Ser Glu Arg Gly Gly Asp Phe
Lys Leu Ile Leu Gly Gln Asp Leu Ser 290 295
300Ile Gly Tyr Glu Asp Arg Glu Lys Asp Ala Val Arg Leu Phe Ile
Thr305 310 315 320Glu Thr
Phe Thr Phe Gln Val Val Asn Pro Glu Ala Leu Ile Leu Leu
325 330 335Lys Phe Ser Gly Gly Ser
34052342PRTArtificial SequenceSynthetic polypeptide 52Met Lys Trp Lys
Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1 5
10 15Val Val Thr Thr Ala Lys Lys Pro Leu Ile
Val Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Gly Gly Gly Thr Ser Glu Phe Leu Lys Arg Ser Phe Ala
35 40 45Pro Leu Thr Glu Lys Gln Trp Gln
Glu Ile Asp Asn Arg Ala Arg Glu 50 55
60Ile Phe Lys Thr Gln Leu Tyr Gly Arg Lys Phe Val Asp Val Glu Gly65
70 75 80Pro Tyr Gly Trp Glu
Tyr Ala Ala His Pro Leu Gly Glu Val Glu Val 85
90 95Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Gly
Leu Ser Asp Glu Asn 100 105
110Glu Val Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu
115 120 125Arg Ala Thr Phe Thr Leu Asp
Leu Trp Glu Leu Asp Asn Leu Glu Arg 130 135
140Gly Lys Pro Asn Val Asp Leu Ser Ser Leu Glu Glu Thr Val Arg
Lys145 150 155 160Val Ala
Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser
165 170 175Gly Val Lys Gly Leu Leu Ser
Phe Glu Glu Arg Lys Gly Gly Gly Gly 180 185
190Gly Glu Asn Leu Tyr Phe Gln Gly His His His His His His
Gly Gly 195 200 205Gly Gly Gly Ile
Glu Cys Gly Ser Thr Pro Lys Asp Leu Leu Glu Ala 210
215 220Ile Val Arg Ala Leu Ser Ile Phe Ser Lys Asp Gly
Ile Glu Gly Pro225 230 235
240Tyr Thr Leu Val Ile Asn Thr Asp Arg Trp Ile Asn Phe Leu Lys Glu
245 250 255Glu Ala Gly His Tyr
Pro Leu Glu Lys Arg Val Glu Glu Cys Leu Arg 260
265 270Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu Asp
Ala Leu Val Val 275 280 285Ser Glu
Arg Gly Gly Asp Phe Lys Leu Ile Leu Gly Gln Asp Leu Ser 290
295 300Ile Gly Tyr Glu Asp Arg Glu Lys Asp Ala Val
Arg Leu Phe Ile Thr305 310 315
320Glu Thr Phe Thr Phe Gln Val Val Asn Pro Glu Ala Leu Ile Leu Leu
325 330 335Lys Phe Ser Gly
Gly Ser 34053319PRTArtificial SequenceSynthetic polypeptide
53Met Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1
5 10 15Val Val Thr Thr Ala Lys
Lys Pro Leu Ile Val Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Gly Gly Gly Thr Ser Glu Phe Leu Lys Arg
Ser Phe Ala 35 40 45Pro Leu Thr
Glu Lys Gln Trp Gln Glu Ile Asp Asn Arg Ala Arg Glu 50
55 60Ile Phe Lys Thr Gln Leu Tyr Gly Arg Lys Phe Val
Asp Val Glu Gly65 70 75
80Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro Leu Gly Glu Val Glu Val
85 90 95Leu Ser Asp Glu Asn Glu
Val Val Lys Trp Gly Leu Arg Lys Gly Gly 100
105 110Glu Asn Leu Tyr Phe Gln Gly Gly Gly Ser Leu Pro
Leu Ile Glu Leu 115 120 125Arg Ala
Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg 130
135 140Gly Lys Pro Asn Val Asp Leu Ser Ser Leu Glu
Glu Thr Val Arg Lys145 150 155
160Val Ala Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser
165 170 175Gly Val Lys Gly
Leu Leu Ser Phe Glu Glu Arg Lys Ile Glu Cys Gly 180
185 190Ser Thr Pro Lys Asp Leu Leu Glu Ala Ile Val
Arg Ala Leu Ser Ile 195 200 205Phe
Ser Lys Asp Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr 210
215 220Asp Arg Trp Ile Asn Phe Leu Lys Glu Glu
Ala Gly His Tyr Pro Leu225 230 235
240Glu Lys Arg Val Glu Glu Cys Leu Arg Gly Gly Lys Ile Ile Thr
Thr 245 250 255Pro Arg Ile
Glu Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe 260
265 270Lys Leu Ile Leu Gly Gln Asp Leu Ser Ile
Gly Tyr Glu Asp Arg Glu 275 280
285Lys Asp Ala Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln Val 290
295 300Val Asn Pro Glu Ala Leu Ile Leu
Leu Lys Phe Ser Gly Gly Ser305 310
31554325PRTArtificial SequenceSynthetic polypeptide 54Met Glu Phe Leu Lys
Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp1 5
10 15Gln Glu Ile Asp Asn Arg Ala Arg Glu Ile Phe
Lys Thr Gln Leu Tyr 20 25
30Gly Arg Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala
35 40 45Ala His Pro Leu Gly Glu Val Glu
Val Leu Ser Asp Glu Asn Glu Val 50 55
60Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu Arg Ala65
70 75 80Thr Phe Thr Leu Asp
Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys 85
90 95Pro Asn Val Asp Leu Ser Ser Leu Glu Glu Thr
Val Arg Lys Val Ala 100 105
110Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser Gly Val
115 120 125Lys Gly Leu Leu Ser Phe Glu
Glu Arg Lys Gly Gly Gly Gly Gly Glu 130 135
140Asn Leu Tyr Phe Gln Gly His His His His His His Gly Gly Gly
Gly145 150 155 160Gly Ile
Glu Cys Gly Ser Thr Pro Lys Asp Leu Leu Glu Ala Ile Val
165 170 175Arg Ala Leu Ser Ile Phe Ser
Lys Asp Gly Ile Glu Gly Pro Tyr Thr 180 185
190Leu Val Ile Asn Thr Asp Arg Trp Ile Asn Phe Leu Lys Glu
Glu Ala 195 200 205Gly His Tyr Pro
Leu Glu Lys Arg Val Glu Glu Cys Leu Arg Gly Gly 210
215 220Lys Ile Ile Thr Thr Pro Arg Ile Glu Asp Ala Leu
Val Val Ser Glu225 230 235
240Arg Gly Gly Asp Phe Lys Leu Ile Leu Gly Gln Asp Leu Ser Ile Gly
245 250 255Tyr Glu Asp Arg Glu
Lys Asp Ala Val Arg Leu Phe Ile Thr Glu Thr 260
265 270Phe Thr Phe Gln Val Val Asn Pro Glu Ala Leu Ile
Leu Leu Lys Phe 275 280 285Ser Gly
Gly Ser Glu Asn Leu Tyr Phe Gln Gly Lys Trp Lys Ser Phe 290
295 300Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys Val
Val Thr Thr Ala Lys305 310 315
320Lys Pro Leu Ile Val 32555157PRTArtificial
SequenceSynthetic polypeptide 55Met Lys Trp Lys Ser Phe Ile Lys Lys Leu
Thr Lys Ala Ala Lys Lys1 5 10
15Val Val Thr Thr Ala Lys Lys Pro Leu Ile Val Glu Asn Leu Tyr Phe
20 25 30Gln Gly Gly Thr Met Ser
Asp Lys Ile Ile His Leu Thr Asp Asp Ser 35 40
45Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val
Asp Phe 50 55 60Trp Ala Glu Trp Cys
Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp65 70
75 80Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu
Thr Val Ala Lys Leu Asn 85 90
95Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile
100 105 110Pro Thr Leu Leu Leu
Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 115
120 125Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu
Asp Ala Asn Leu 130 135 140Ala Gly Ser
Gly Ser Gly Thr His His His His His His145 150
15556144PRTArtificial SequenceSynthetic polypeptide 56Met Lys Trp
Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1 5
10 15Val Val Thr Thr Ala Lys Lys Pro Leu
Ile Val Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Thr Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro
35 40 45Glu Val Lys Pro Glu Val Lys
Pro Glu Thr His Ile Asn Leu Lys Val 50 55
60Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro65
70 75 80Leu Arg Arg Leu
Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met 85
90 95Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile
Arg Ile Gln Ala Asp Gln 100 105
110Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His
115 120 125Arg Glu Gln Ile Gly Gly Gly
Ser Leu Glu His His His His His His 130 135
14057264PRTArtificial SequenceSynthetic polypeptide 57Met Lys Trp
Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys Lys1 5
10 15Val Val Thr Thr Ala Lys Lys Pro Leu
Ile Val Glu Asn Leu Tyr Phe 20 25
30Gln Gly Gly Thr Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly
35 40 45Leu Val Gln Pro Thr Arg Leu
Leu Leu Glu Tyr Leu Glu Glu Lys Tyr 50 55
60Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys65
70 75 80Lys Phe Glu Leu
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp 85
90 95Gly Asp Val Lys Leu Thr Gln Ser Met Ala
Ile Ile Arg Tyr Ile Ala 100 105
110Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile
115 120 125Ser Met Leu Glu Gly Ala Val
Leu Asp Ile Arg Tyr Gly Val Ser Arg 130 135
140Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu
Ser145 150 155 160Lys Leu
Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys
165 170 175Thr Tyr Leu Asn Gly Asp His
Val Thr His Pro Asp Phe Met Leu Tyr 180 185
190Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu
Asp Ala 195 200 205Phe Pro Lys Leu
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln 210
215 220Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala
Trp Pro Leu Gln225 230 235
240Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Gly Ser
245 250 255Leu Glu His His His
His His His 26058413PRTArtificial SequenceSynthetic
polypeptide 58Met Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Lys Ala Ala Lys
Lys1 5 10 15Val Val Thr
Thr Ala Lys Lys Pro Leu Ile Val Glu Asn Leu Tyr Phe 20
25 30Gln Gly Gly Thr Met Lys Ile Glu Glu Gly
Lys Leu Val Ile Trp Ile 35 40
45Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe 50
55 60Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu His Pro Asp Lys Leu65 70 75
80Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro
Asp Ile 85 90 95Ile Phe
Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu 100
105 110Leu Ala Glu Ile Thr Pro Asp Lys Ala
Phe Gln Asp Lys Leu Tyr Pro 115 120
125Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro
130 135 140Ile Ala Val Glu Ala Leu Ser
Leu Ile Tyr Asn Lys Asp Leu Leu Pro145 150
155 160Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu
Asp Lys Glu Leu 165 170
175Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr
180 185 190Phe Thr Trp Pro Leu Ile
Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr 195 200
205Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn
Ala Gly 210 215 220Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His225 230
235 240Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu Ala Ala Phe Asn Lys 245 250
255Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile
260 265 270Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys 275
280 285Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser
Ala Gly Ile Asn 290 295 300Ala Ala Ser
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr305
310 315 320Leu Leu Thr Asp Glu Gly Leu
Glu Ala Val Asn Lys Asp Lys Pro Leu 325
330 335Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu
Val Lys Asp Pro 340 345 350Arg
Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro 355
360 365Asn Ile Pro Gln Met Ser Ala Phe Trp
Tyr Ala Val Arg Thr Ala Val 370 375
380Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp385
390 395 400Ala Gln Thr Gly
Ser Leu Glu His His His His His His 405
4105928PRTT. maritima 59Tyr Ala Ala His Pro Leu Gly Glu Val Glu Val Leu
Ser Asp Glu Asn1 5 10
15Glu Val Val Lys Trp Gly Leu Arg Lys Ser Leu Pro 20
256028PRTArtificial SequenceSynthetic polypeptide 60Tyr Thr Val
Val Pro Glu Gly Arg Leu Lys Lys Ile Glu Asp Asn Pro1 5
10 15Gly Asn Val Cys Thr Gly Met Tyr Gln
Val Lys Pro 20 256128PRTArtificial
SequenceSynthetic polypeptide 61Tyr Ala Ala Val Asn Thr Gly Glu Leu Arg
Pro Ile Asp Asp Thr Pro1 5 10
15Glu Asp Val Asp Met Lys Leu Arg Gln Val Gln Pro 20
256228PRTArtificial SequenceSynthetic polypeptide 62Tyr Ala
Ala Val Asn Thr Gly Arg Arg Thr Ala Leu Glu Asp Lys Ala1 5
10 15Glu Gly Ala Ser Ile Phe Gln Arg
Gln Val Leu Pro 20 256328PRTArtificial
SequenceSynthetic polypeptide 63Phe Ser Ala Leu Gly Thr Gly His Val Ser
Arg Val Ala Ala Asp Thr1 5 10
15Pro Gly Val Glu Ala Leu Gln Arg His Val Val Arg 20
256488PRTT. maritima 64Leu Leu Ser Phe Glu Glu Arg Lys Ile
Glu Cys Gly Ser Thr Pro Lys1 5 10
15Asp Leu Leu Glu Ala Ile Val Arg Ala Leu Ser Ile Phe Ser Lys
Asp 20 25 30Gly Ile Glu Gly
Pro Tyr Thr Leu Val Ile Asn Thr Asp Arg Trp Ile 35
40 45Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro Leu
Glu Lys Arg Val 50 55 60Glu Glu Cys
Leu Arg Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu65 70
75 80Asp Ala Leu Val Val Ser Glu Arg
856588PRTGluconacetobacter diazotrophicus 65Leu Arg Glu Gly
Thr Ser Asn Pro Lys Leu Ala Leu Pro Ser Ser Ala1 5
10 15Ser Asp Tyr Pro Ala Ala Ile Ala Ala Ala
Leu Asn Gln Leu Arg Leu 20 25
30Ala Gly Val Asn Gly Pro Tyr Ala Val Val Leu Gly Ala Gly Val Tyr
35 40 45Thr Ala Leu Ser Gly Gly Asp Asp
Glu Gly Tyr Pro Val Phe Arg His 50 55
60Ile Glu Ser Leu Ile Asp Gly Lys Ile Val Trp Ala Pro Ala Ile Glu65
70 75 80Gly Gly Phe Val Leu
Ser Thr Arg 856693PRTHungateiclostridium thermocellum
66Leu Leu Thr Glu Asp Gly Ile Val Lys Phe Pro Ile Ser Asn Trp Ser1
5 10 15Glu Gly Glu Asn Pro Phe
Lys Asp Ile Ser Ile Gly Leu Ala Lys Phe 20 25
30Ile Glu Asn Gly Ile Val Gly Arg Lys Ala Leu Val Val
Ser Pro Asn 35 40 45Leu Phe Val
Gln Leu Gln Arg Ile Gln Pro Gly Thr Gly Thr Thr Glu 50
55 60Tyr Asp Arg Ile Asn Lys Leu Leu Asp Gly Asn Ile
Phe Ser Thr Pro65 70 75
80Val Leu Lys Asp Asp Lys Ala Val Leu Val Cys Ser Glu 85
906792PRTBrachyspira hyodysenteriae 67Ile Leu Asn Ala Glu
Gly Ala Gln Lys Leu Gln Ile Ser Asp Trp Gly1 5
10 15Gln Gly Glu Asn Pro Tyr Thr Asp Ile Val Lys
Ala Ile Asn Met Ile 20 25
30Arg Glu Lys Gly Ile Val Gly Arg Phe Val Leu Cys Leu Ser Gln Ser
35 40 45Leu Tyr Phe Asp Leu Gln Arg Ile
Gln Gln Gly Thr Gly Met Thr Glu 50 55
60Ala Gln Arg Ile Ser Ser Met Ile Gly Asn Leu Tyr Asn Val Pro Val65
70 75 80Ile Lys Gly Lys Lys
Ala Ala Leu Ile Cys Ala Glu 85
906893PRTEggerthella 68Leu Leu Thr Val Lys Gly Ser Ser Lys Ile Lys Lys
Ser Asp Trp Ser1 5 10
15Gln Gly Glu Asn Ser Phe Ala Asp Ile Thr Ala Gly Val Ala Gln Leu
20 25 30Ala Lys Thr Gly Tyr Leu Gly
Arg Tyr Ala Leu Val Val Ser Pro Asp 35 40
45Leu Phe Leu Asp Leu Gln Arg Leu Gln Pro Asn Thr Gly Leu Leu
Glu 50 55 60Ile Asp Arg Ile Lys Lys
Leu Ile Gly Asp Asn Val Tyr Met Thr Ser65 70
75 80Val Met Gly Pro Gly Lys Ala Val Leu Val Cys
Ala Glu 85 90697PRTArtificial
SequenceSynthetic polypeptide 69Glu Asn Leu Tyr Phe Gln Gly1
5707PRTArtificial SequenceSynthetic polypeptidemisc_featurewherein X = S
or Gmisc_feature(7)..(7)Xaa can be any naturally occurring amino acid
70Glu Asn Leu Tyr Phe Gln Xaa1 5715PRTArtificial
SequenceSynthetic polypeptide 71Asp Asp Asp Asp Lys1
5727PRTArtificial SequenceSynthetic polypeptide 72Glu Asn Leu Tyr Phe Gln
Gly1 5738PRTArtificial SequenceSynthetic polypeptide 73Leu
Glu Val Leu Phe Gln Gly Pro1 5746PRTArtificial
SequenceSynthetic polypeptide 74Leu Val Pro Arg Gly Ser1
5755PRTArtificial SequenceSynthetic polypeptide 75Gly Gly Gly Gly Ser1
5765PRTArtificial SequenceSynthetic polypeptide 76Gly Ser Gly
Ser Gly1 5774PRTArtificial SequenceSynthetic polypeptide
77Gly Gly Gly Gly1783PRTArtificial SequenceSynthetic polypeptide 78Gly
Gly Gly1792PRTArtificial SequenceSynthetic polypeptide 79Gly
Gly1802PRTArtificial SequenceSynthetic polypeptide 80Gly
Ser1814PRTArtificial SequenceSynthetic polypeptide 81Gly Ser Gly
Ser1824PRTArtificial SequenceSynthetic polypeptide 82Gly Gly Gly
Ser1833PRTArtificial SequenceSynthetic polypeptide 83Gly Gly
Ser1843PRTArtificial SequenceSynthetic polypeptide 84Gly Thr
Ser1855PRTArtificial SequenceSynthetic polypeptide 85Gly Gly Gly Gly Thr1
586265PRTStigmatella aurantiaca DW4/3?1 86Pro Asp Phe Leu
Gly His Ala Glu Asn Pro Leu Arg Glu Glu Glu Trp1 5
10 15Ala Arg Leu Asn Glu Thr Val Ile Gln Val
Ala Arg Arg Ser Leu Val 20 25
30Gly Arg Arg Ile Leu Asp Ile Tyr Gly Pro Leu Gly Ala Gly Val Gln
35 40 45Ser Val Pro His Asp Glu Tyr Gln
Gly Val Ser Ser Gly Ala Ile Asp 50 55
60Ile Val Gly Glu Gln Glu Thr Ala Thr Val Phe Thr Asp Val Arg Lys65
70 75 80Phe Lys Thr Ile Pro
Ile Ile Tyr Lys Asp Phe Leu Leu His Trp Arg 85
90 95Asp Ile Glu Ala Ala Arg Ile His Asn Met Pro
Leu Asp Val Ser Ala 100 105
110Ala Ala Gly Ala Ala Ala Leu Cys Ala Gln Gln Glu Asp Glu Leu Ile
115 120 125Phe Tyr Gly Asp Pro Lys Leu
Gly His Glu Gly Leu Met Thr Ala Thr 130 135
140Asp Arg Leu Thr Val Pro Leu Gly Asp Trp Ala Thr Pro Gly Ala
Gly145 150 155 160Tyr Val
Ala Ile Val Glu Ala Thr Arg Lys Leu Asn Glu His Gly His
165 170 175Tyr Gly Pro Tyr Ala Val Val
Leu Ser Pro Arg Leu Tyr Ser Leu Leu 180 185
190His Arg Ile Phe Glu Lys Thr Gly Val Leu Glu Ile Glu Thr
Ile Arg 195 200 205Gln Leu Ala Ser
Asp Gly Val Phe Gln Ser Asn Arg Leu Arg Gly Asp 210
215 220Ser Gly Val Val Val Ser Thr Gly Arg Glu Asn Met
Asp Leu Thr Val225 230 235
240Ala Met Asp Met Val Thr Ala Tyr Leu Gly Ala Ser Arg Met Asn His
245 250 255Pro Phe Arg Val Leu
Glu Ala Leu Ile 260 26587258PRTRhodospirillum
rubrum ATCC 11170 87Met Asn Asp Leu Met Arg Asp Leu Ala Pro Ile Ser Ala
Lys Ala Trp1 5 10 15Ala
Glu Ile Glu Thr Glu Ala Arg Gly Thr Leu Thr Val Thr Leu Ala 20
25 30Ala Arg Lys Val Val Asp Phe Lys
Gly Pro Leu Gly Trp Asp Ala Ser 35 40
45Ser Val Ser Leu Gly Arg Thr Glu Ala Leu Ala Glu Glu Pro Lys Ala
50 55 60Ala Gly Ser Ala Ala Val Val Thr
Val Arg Lys Arg Ala Val Gln Pro65 70 75
80Leu Ile Glu Leu Cys Val Pro Phe Thr Leu Lys Arg Ala
Glu Leu Glu 85 90 95Ala
Ile Ala Arg Gly Ala Ser Asp Ala Asp Leu Asp Pro Val Ile Glu
100 105 110Ala Ala Arg Ala Ile Ala Ile
Ala Glu Asp Arg Ala Val Phe His Gly 115 120
125Phe Ala Ala Gly Gly Ile Thr Gly Ile Gly Glu Ala Ser Ala Glu
His 130 135 140Ala Leu Asp Leu Pro Ala
Asp Leu Ala Asp Phe Pro Gly Val Leu Val145 150
155 160Arg Ala Leu Ala Val Leu Arg Asp Arg Gly Val
Asp Gly Pro Tyr Ala 165 170
175Leu Val Leu Gly Arg Thr Val Tyr Gln Gln Leu Met Glu Thr Thr Thr
180 185 190Pro Gly Gly Tyr Pro Val
Leu Gln His Val Arg Arg Leu Phe Glu Gly 195 200
205Pro Leu Ile Trp Ala Pro Gly Val Asp Gly Ala Met Leu Ile
Ser Gln 210 215 220Arg Gly Gly Asp Phe
Glu Leu Thr Val Gly Arg Asp Phe Ser Ile Gly225 230
235 240Tyr His Asp His Asp Ala Gln Ser Val His
Leu Tyr Leu Gln Glu Ser 245 250
255Met Thr88253PRTFrankia alni ACN14a 88Met Asn His Leu Leu Arg Gly
His Ala Pro Leu Thr Asp Ala Ala Trp1 5 10
15Lys Ala Val Asp Asp Glu Ala Lys Ala Arg Leu Thr Thr
Asn Leu Ala 20 25 30Ala Arg
Lys Val Val Asp Phe Ala Gly Pro His Gly Trp Glu Tyr Ser 35
40 45Ala Thr Ala Leu Gly Arg Val Ala Ala Leu
Ser Ala Pro Pro Ala Ala 50 55 60Gly
Val Gln Ala Arg Val Arg Gln Val Gln Pro Val Ile Glu Leu Arg65
70 75 80Val Gly Phe Thr Leu Asp
Arg Ala Glu Leu Ala Asp Ala Asp Arg Gly 85
90 95Ala Asp Asp Leu Asp Leu Ala Pro Leu Glu Glu Ala
Val Arg Arg Ile 100 105 110Ala
Val Thr Glu Asn Ser Val Val Phe His Gly Tyr Gln Glu Ala Gly 115
120 125Leu Val Gly Ile Thr Gln Ala Ser Ser
His Pro Gln Leu Thr Leu Glu 130 135
140Ala Gly Thr Asp Thr Tyr Pro Arg Thr Val Ala Lys Ala Val Ala Leu145
150 155 160Leu Arg Arg Ala
Gly Ile Ala Gly Pro Tyr Ala Leu Ala Leu Glu Pro 165
170 175Asp Ser Tyr Thr Ala Val Ile Glu Thr Ala
Glu His Gly Gly Tyr Leu 180 185
190Leu Leu Thr His Leu Gln His Ile Leu Asp Gly Pro Val Val Gln Ala
195 200 205Pro Gly Val Thr Gly Ala Val
Val Leu Ser Leu Arg Gly Gly Asp Phe 210 215
220Val Leu Glu Ser Gly Gln Asp Leu Ser Ile Gly Tyr Ala Ser His
Thr225 230 235 240Ala Asp
Thr Val Asp Leu Tyr Leu Glu Glu Ser Phe Thr 245
25089251PRTRhodococcus jostii RHA1 89Ser Ser Asn Leu His Arg Asn Leu
Ala Pro Val Thr Glu Val Ala Trp1 5 10
15Gln Gln Ile Gly Glu Glu Ala Ala Arg Thr Phe Lys Arg His
Val Ala 20 25 30Gly Arg Arg
Val Val Asp Val Ala Gly Pro Phe Gly Tyr Ser Tyr Ser 35
40 45Ala His Asn Leu Gly Arg Val Thr Pro Ile Lys
Thr Ser Asp Ser Arg 50 55 60Ile Arg
Ala Gln Gln Arg Gln Val Asn Pro Leu Val Glu Leu Arg Phe65
70 75 80Pro Phe Thr Leu Ser Arg Ala
Glu Val Asp Asp Val Ala Arg Gly Ser 85 90
95Leu Asp Ser Asp Trp Gln Pro Val Lys Asp Ala Ala Lys
Ala Val Ala 100 105 110Phe Ala
Glu Asp Gln Ser Ile Phe Gln Gly Phe Asp Glu Ala Gly Ile 115
120 125Arg Gly Leu Gly Pro Ser Ser Asp Asn Pro
Val Leu Ser Leu Pro Glu 130 135 140Asp
Pro Leu Leu Ile Pro Asp Ala Val Ala Ser Ala Leu Ser Ala Leu145
150 155 160Arg Leu Ala Gly Val Glu
Gly Pro Tyr Ser Val Val Leu Asp Ala Asp 165
170 175Ala Tyr Thr Ala Val Ser Glu Thr Arg Asp Glu Gly
His Pro Val Phe 180 185 190His
His Leu Arg Asp Leu Val Ala Gly Asp Ile Ile Trp Ala Pro Ala 195
200 205Ile Ser Gly Gly Tyr Val Leu Ser Thr
Arg Gly Gly Asp Asn Gln Leu 210 215
220Thr Leu Gly Thr Asp Leu Ser Ile Gly Tyr Asp Ser His Thr Ala Thr225
230 235 240Asp Val Thr Leu
Tyr Leu Glu Glu Thr Phe Thr 245
25090252PRTNocardia farcinica 90Met Asn Asn Leu His Arg Glu Leu Ala Pro
Ile Thr Ser Glu Ala Trp1 5 10
15Ala Ala Ile Glu Glu Glu Ala Gly Arg Thr Phe Lys Arg His Ile Ala
20 25 30Gly Arg Arg Val Val Asp
Val Ala Gly Pro His Gly Val Asp Phe Ser 35 40
45Ala Val Gly Leu Gly Arg Thr Thr Gly Ile Ala Ala Pro Asp
Glu Gly 50 55 60Val Gln Ala Arg Gln
Arg Val Val Ala Pro Leu Val Glu Leu Arg Val65 70
75 80Pro Phe Thr Leu Ser Arg Glu Glu Leu Asp
Asn Val Glu Arg Gly Ala 85 90
95Lys Asp Thr Asp Leu Asp Ala Val Lys Glu Ala Ala Arg Arg Ile Ala
100 105 110Phe Ala Glu Asp Arg
Ala Ile Phe Glu Gly Tyr Pro Ala Ala Gly Ile 115
120 125Thr Gly Ile Arg Ala Ala Gly Ser Asn Ala Pro Ile
Thr Val Pro Asp 130 135 140Asp Ala Arg
Leu Val Pro Glu Ala Ile Thr Gln Ala Leu Thr Ala Leu145
150 155 160Arg Leu Ala Gly Val Asp Gly
Pro Tyr Ser Val Leu Leu Ser Ala Glu 165
170 175Leu Tyr Thr Glu Val Ser Glu Thr Ser Asp His Gly
Tyr Pro Ile Arg 180 185 190Thr
His Ile Glu Arg Leu Ile Pro Asp Gly Glu Ile Ile Trp Ala Pro 195
200 205Ala Ile Asp Gly Ala Phe Val Leu Thr
Thr Arg Gly Gly Asp Tyr Glu 210 215
220Leu Thr Leu Gly Gln Asp Val Ser Ile Gly Tyr Leu Ser His Asp Ala225
230 235 240Asp Thr Val Arg
Leu Tyr Phe Gln Gln Thr Met Gln 245
250919PRTArtificial SequenceSynthetic polypeptidemisc_feature(1)..(9)X1,
X2, X3, X4, X5, X6, X7, X8, X9 are any amino acid forming N-terminal
of the 1st fragment P-domain in an engineered microcompartment
protein 91Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
59213PRTArtificial SequenceSynthetic polypeptidemisc_feature(1)..(9)X1,
X2, X3, X4, X5, X6, X7, X8, X9 are any amino acid forming the
N-terminal of the 1st fragment P-domain in an engineered
microcompartment proteinmisc_feature(10)..(13)Xaa can be any naturally
occurring amino acidmisc_feature(100)..(103)X100, X101, X102, X103 are
any amino acid forming an inserted target peptide 92Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
109314PRTArtificial SequenceSynthetic polypeptidemisc_feature(1)..(14)Xaa
can be any naturally occurring amino acidmisc_feature(65)..(78)X65, X66,
X67, X68, X69, X70, X71, X72, X73, X74, X75, X76, X77, X78 are any
amino acid forming the C-terminal of an E-loop in an engineered
microcompartment protein 93Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 109414PRTArtificial
SequenceSynthetic polypeptidemisc_feature(1)..(14)Xaa can be any
naturally occurring amino acidmisc_feature(131)..(144)X131, X132, X133,
X134, X135, X136, X137, X138, X139, X140, X141, X142, X143,
X144 are any amino acid forming the N-terminal of an A-Domain in an
engineered microcompartment protein 94Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5
1095252PRTWolinella succinogenes 95Met Asp Ile Leu Arg Arg Glu Asn Ala
Gln Phe Pro Ala Ser Ile Trp1 5 10
15Ser Ala Ile Glu Lys Glu Ala Gly Leu Val Phe Gly Lys His Leu
Thr 20 25 30Gly Arg Lys Val
Val Asp Phe Lys Gly Gly Leu Gly Ile Gly Phe Ser 35
40 45Ser Leu Pro Thr Gly Arg Val Ile Ser Ser Lys Glu
Lys Leu Gly Glu 50 55 60Ala Ser Val
Gly Val Arg Met Asn Thr Pro Val Ile Glu Leu Lys Ile65 70
75 80Pro Phe Ser Phe Pro Glu Ser Glu
Val Glu Ala Ile Leu Arg Glu Ala 85 90
95Asn Ala Phe Asp Ile Ser Ser Ile Glu Lys Ala Ala Lys Lys
Val Cys 100 105 110Val Ala Glu
Asn Glu Leu Val Phe Tyr Gly Leu Lys Lys Glu Gly Ile 115
120 125Glu Gly Leu Ile Pro Ser Ile Pro His Lys Pro
Ile Lys Ala Lys Gly 130 135 140Asp Glu
Ile Leu Pro Ala Val Ala Glu Gly Ile Lys Glu Leu Val Asn145
150 155 160Ser Glu Ile Glu Gly Pro Tyr
Ala Leu Leu Ile Gln Pro Gln Tyr Phe 165
170 175Gly Lys Leu Phe Gly Val Ala Gly Asn Ser Gly Tyr
Pro Leu Thr Leu 180 185 190Lys
Leu Ala Glu Leu Leu Gln Gly Asn Asn Ile Ile Val Ala Pro Ala 195
200 205Leu Lys Ser Gly Ala Leu Leu Val Ser
Leu Arg Gly Gly Asp Tyr Glu 210 215
220Leu Tyr Ser Gly Met Asp Ile Gly Val Gly Tyr Ser Glu Lys Lys Ser225
230 235 240Thr Asn His Glu
Leu Phe Phe Phe Glu Thr Leu Thr 245
25096234PRTSaccharolobus solfataricus 96Glu Thr Lys Asp Phe Pro Leu Ile
Pro Thr Ser Ser Lys Glu Ile Ser1 5 10
15Lys Asp His Ile Val Ser Trp Ile Thr Glu Gly Ile Val Ser
Ser Arg 20 25 30Ile Met Arg
Asn Ile Gly Asn Thr Ile Lys Tyr Glu Phe Thr Thr Ile 35
40 45Pro Leu Ser Glu Ile Lys Glu Asp Ser Gly Asp
Ile Ile Gln Ser Lys 50 55 60Ser Ala
Ser Leu Tyr Glu Val Pro Leu Ile Asn Ser Gln Val Lys Phe65
70 75 80Tyr Leu Gly Gln Lys Ser Asp
Ser Arg Arg Thr Ala Val Leu Ala Gly 85 90
95Lys Ser Phe Ala Lys Met Glu Asn Tyr Leu Leu Leu Lys
Asn His Pro 100 105 110Leu Ser
Pro Leu Lys Ile Gly Leu Lys Ile Thr Gly Ser Asp Trp Asn 115
120 125Val Ala Gly Asn Ile Leu Leu Asp Val Leu
Arg Ala Tyr Glu Asn Leu 130 135 140Thr
Arg Glu Gly Phe Gly Lys Asp Val Tyr Ile Leu Met Ser Ser Leu145
150 155 160Asn Tyr Ser Lys Thr Phe
Arg Val Val Asp Arg Ser Gly Thr Tyr Glu 165
170 175Ile Glu Met Ile Lys Glu Ile Gly Asn Val Val Pro
Thr Asp Ile Val 180 185 190Ser
Asn Asp Glu Ile Tyr Val Ile Ser Lys Gln Gly Phe Asp Ile Leu 195
200 205Val Phe Ser Asp Leu Asn Val Glu Tyr
Leu Ser Lys Glu Lys Asp Tyr 210 215
220Glu Val Tyr Leu Ile Thr Glu Gln Ile Ala225
23097265PRTGeobacillus kaustophilus 97Met Asp Lys Thr Lys Leu Tyr Pro Glu
Ala Pro Leu Thr Ser Ser Gln1 5 10
15Trp Gly Glu Leu Asp Glu Leu Val Ile Glu Thr Ala Arg Arg Gln
Leu 20 25 30Val Gly Arg Arg
Phe Ile Asp Leu Tyr Gly Pro Leu Gly Glu Gly Val 35
40 45Gln Ser Val Ala Asn Asp Ile Tyr Met Asn Pro Glu
Gln Gly Asp Met 50 55 60Ser Phe His
Gly Lys Glu Leu Ser Leu Ser Glu Pro Ala Arg Arg Val65 70
75 80His Leu Thr Ile Pro Leu Leu Tyr
Lys Asp Phe Ile Leu Tyr Trp Arg 85 90
95Asp Ile Glu Gln Ala Lys Gln Leu Gly Ser Pro Ile Asp Phe
Ser Ala 100 105 110Ala Ala Asn
Ala Ala Gln Gln Cys Ala Leu Leu Glu Asp Asp Leu Ile 115
120 125Phe Asn Gly Ser Thr Glu Phe Asp Val Pro Gly
Ile Met Asn Val Lys 130 135 140Gly Lys
Ile Ala His Ile Arg Ser Asp Trp Met Lys Ser Gly Asn Ala145
150 155 160Phe Thr Asp Val Val Glu Ala
Arg Asn Lys Leu Leu Gln Leu Gly His 165
170 175Thr Gly Pro Tyr Ala Leu Val Leu Ser Pro Glu Leu
Tyr Ala Leu Ile 180 185 190His
Arg Val His Glu Gly Thr His Val Leu Glu Ile Glu His Ile Arg 195
200 205Glu Leu Met Thr Ala Gly Ile Tyr Gln
Thr Pro Val Ile Lys Gly Lys 210 215
220Arg Gly Val Val Ile Asp Thr Gly Arg Gln Asn Ile Asp Leu Ala Val225
230 235 240Ala Val Asp Val
Gln Thr Ala Phe Leu Asp Thr Glu Asn Met Asn Tyr 245
250 255Leu Phe Arg Val Tyr Glu Ser Val Val
260 26598267PRTAquifex aeolicus 98Met Glu Phe Leu
Gln Arg Asp Gln Ala Pro Leu Thr Ala Glu Glu Trp1 5
10 15Glu Gln Ile Asp Lys Thr Ala Tyr Glu Val
Phe Lys Ser Thr Val Val 20 25
30Cys Arg Lys Phe Met Pro Val Val Gly Pro Phe Gly Ala Gly His Gln
35 40 45Val Val Ser Tyr Asp Val Leu Tyr
Gly Val Glu Pro Gly Val Cys Glu 50 55
60Val Lys Pro Gly Gln Glu Tyr Lys Val Cys Glu Pro Val Arg Thr Gly65
70 75 80Glu Arg Lys His Val
Pro Val Pro Thr Leu Tyr Lys Asp Phe Val Ile 85
90 95Ser Trp Arg Asp Leu Glu His Trp Arg Gln Phe
Asn Leu Pro Val Asp 100 105
110Thr Thr Gly Val Ala Ala Ala Ala Ser Ser Leu Ala Val Ala Glu Asp
115 120 125Lys Leu Ile Leu Phe Gly Asn
Gln Glu Met Gly Ile Glu Gly Phe Leu 130 135
140Thr Ala Lys Gly Thr Leu Arg Glu Glu Leu Ser Asp Trp Glu Lys
Val145 150 155 160Gly Asn
Ala Phe Gln Asp Val Val Lys Gly Ile Ser Arg Leu Val Glu
165 170 175Lys Gly Phe Tyr Thr Asn Tyr
Tyr Leu Ile Val Asn Pro Lys Arg Tyr 180 185
190Phe Leu Leu Asn Arg Ile His Asp Asn Thr Gly Leu Leu Glu
Leu Glu 195 200 205Gln Ile Lys Lys
Val Val Lys Glu Val Tyr Gln Thr Pro Ile Ile Pro 210
215 220Glu Asp Ile Val Leu Leu Val Ser Ala Ser Pro Ala
Asn Phe Asp Leu225 230 235
240Ala Ile Ala Leu Asp Val Asn Val Ala Phe Val Glu Thr Ser Asn Met
245 250 255Asn His Thr Phe Arg
Val Met Glu Met Val Val 260
26599259PRTAnaeromyxobacter dehalogenans 2CP?C 99Met Ser Trp Gln Asp Arg
Asp Gly Ala Pro Phe Gly Gln Gln Val Trp1 5
10 15Asp Arg Ile Asp Glu Ala Ile Glu Ala Ala Ala Ala
Glu Ala Arg Ala 20 25 30Gly
Arg Arg Leu Leu Arg Val Ile Gly Pro Leu Gly Phe Glu Ala Arg 35
40 45Ala Gly Val Ala Asp Asp Ala Pro Ala
Gly Gly Glu Asp Glu Pro Glu 50 55
60Ala Gly Asp Glu Thr His Val His Val Pro Ser Val Arg Ala Leu Pro65
70 75 80Val Leu His Arg Thr
Phe Arg Leu Gly Ala Arg Ala Val Glu Ala Leu 85
90 95Glu Arg Arg Gly Glu Pro Leu Thr Leu Thr Glu
Ala Ala Glu Ala Ala 100 105
110Arg Arg Ile Ala Arg Ala Glu Asp Arg Leu Leu Phe Glu Gly His Ala
115 120 125Gly Ala Gly Val Arg Gly Leu
Leu Glu His Pro Gly Leu Val Glu Val 130 135
140Pro Ala Gly Asp Trp Ala Asp Pro Gly Arg Ala Gly Asp Ala Leu
Leu145 150 155 160Ala Ala
Leu Thr Ala Leu Asp Asp Ala Gly Arg His Gly Pro Tyr Ala
165 170 175Ala Ala Val Ser Pro Ala Arg
Phe Tyr Gln Leu Phe Arg Pro Phe Ala 180 185
190Gly Thr Ala Leu Thr Pro Tyr Gln Gln Leu Leu Pro Ala Phe
Glu Gly 195 200 205Gly Ile Val Lys
Ala Pro Gly Leu Arg Asp Gly Ala Val Val Val Val 210
215 220Arg Ser Ala Ser Gly Pro Gln Ala Val Val Gly Gln
Glu Leu Thr Ala225 230 235
240Ala Tyr Asp Gly Arg Glu Gly Ile Phe His Leu Val Ser Leu Ala Glu
245 250 255Ser Val
Thr100251PRTGluconacetobacter diazotrophicus 100Met Asn Asn Leu His Arg
Glu Leu Ala Pro Ile Ser Glu Ala Ala Trp1 5
10 15Ala Gln Ile Glu Glu Glu Ala Ser Arg Thr Leu Lys
Arg Tyr Leu Ala 20 25 30Ala
Arg Arg Val Val Asp Val Pro Glu Ala Lys Gly Phe Gly Phe Ser 35
40 45Ala Val Gly Thr Gly His Val Glu Arg
Ile Asp Ala Pro Gly Ser Asp 50 55
60Ile Arg Ala Val Arg Arg Asn Val Leu Pro Leu Val Glu Leu Arg Val65
70 75 80Pro Phe Thr Leu Ala
Arg Asp Ala Ile Asp Asp Val Glu Arg Gly Ala 85
90 95Gly Asp Ser Asp Trp Gln Pro Leu Lys Asp Ala
Ala Lys Lys Ile Ala 100 105
110Phe Ala Glu Asp Arg Ala Val Phe Asp Gly Tyr Ala Ala Ala Gly Ile
115 120 125Leu Gly Leu Arg Glu Gly Thr
Ser Asn Pro Lys Leu Ala Leu Pro Ser 130 135
140Ser Ala Ser Asp Tyr Pro Ala Ala Ile Ala Ala Ala Leu Asn Gln
Leu145 150 155 160Arg Leu
Ala Gly Val Asn Gly Pro Tyr Ala Val Val Leu Gly Ala Gly
165 170 175Val Tyr Thr Ala Leu Ser Gly
Gly Asp Asp Glu Gly Tyr Pro Val Phe 180 185
190Arg His Ile Glu Ser Leu Ile Asp Gly Lys Ile Val Trp Ala
Pro Ala 195 200 205Ile Glu Gly Gly
Phe Val Leu Ser Thr Arg Gly Gly Asp Phe Glu Leu 210
215 220Asp Ile Gly Gln Asp Phe Ser Ile Gly Tyr Ser Ser
His Ser Ala Asp225 230 235
240Ser Val Glu Leu Tyr Leu Gln Glu Ser Phe Thr 245
250101251PRTMethylorubrum extorquens 101Met Asn Asn Leu His Arg
Glu Leu Ala Pro Ile Ser Asp Ala Ala Trp1 5
10 15Ala Gln Ile Glu Asp Glu Ala Ser Arg Thr Leu Lys
Arg Tyr Leu Ala 20 25 30Ala
Arg Arg Val Val Asp Val Val Gly Pro Lys Gly Pro Gly Tyr Ala 35
40 45Ala Ala Gly Thr Gly His Thr Arg Pro
Ile Glu Ala Pro Gly Glu Gly 50 55
60Ile Arg Ser Leu Leu Arg Glu Ala Gln Pro Leu Val Glu Leu Arg Val65
70 75 80Pro Phe Thr Leu Thr
Arg Gln Ala Ile Asp Asp Val Glu Arg Gly Ser 85
90 95Glu Asp Ser Asp Trp Gln Pro Leu Lys Asp Ala
Ala Arg Met Leu Ala 100 105
110Phe Ala Glu Asp Arg Ala Val Phe Glu Gly Tyr Ala Ala Ala Gly Ile
115 120 125Gly Gly Ile Gly Lys Gly Ser
Ser Asn Ala Ala Val Pro Leu Pro Ala 130 135
140Thr Leu Asp Asp Tyr Pro Glu Ala Val Ala Arg Ala Leu Asn Asp
Leu145 150 155 160Lys Leu
Ala Gly Cys Asn Gly Pro Tyr Val Leu Val Leu Gly Gly Asp
165 170 175Val Tyr Arg Ala Ala Ser Gly
Gly Asn Glu Glu Gly Tyr Pro Ile Phe 180 185
190His His Leu Glu Arg Ile Val Asp Gly Gly Val Ile Trp Ala
Pro Ala 195 200 205Ile Ala Gly Gly
Phe Val Leu Thr Thr Arg Gly Gly Asp Phe Glu Leu 210
215 220Asp Ile Gly Gln Asp Ile Ser Ile Gly Tyr Leu Ser
His Ser Ala Thr225 230 235
240Thr Val Glu Leu Tyr Leu Gln Glu Ser Phe Thr 245
250102250PRTAlkaliphilus metalliredigens 102Met Asp Ile Leu Lys
Arg Asp Met Ala Pro Leu Thr Glu Ser Val Trp1 5
10 15Glu Glu Ile Asp Gln Arg Ala Ala Glu Val Leu
Lys Thr His Leu Ser 20 25
30Ala Arg Arg Val Val Asn Ile Val Gly Pro Lys Gly Trp Asp Tyr Thr
35 40 45Val Val Pro Glu Gly Arg Leu Lys
Lys Ile Glu Asp Asn Pro Gly Asn 50 55
60Val Cys Thr Gly Met Tyr Gln Val Lys Pro Leu Val Glu Ala Arg Ile65
70 75 80Ser Phe Lys Leu Asp
Arg Trp Glu Met Asp Asn Leu Ile Arg Gly Ala 85
90 95Lys Asp Ile Lys Leu Asp Ala Leu Glu Glu Ala
Ala Glu Lys Met Ala 100 105
110Ile Phe Glu Glu Asn Met Leu Tyr Asn Gly Tyr Lys Pro Gly Asp Ile
115 120 125Glu Gly Leu Ile Glu Ala Ser
Ser His Lys Leu Ser Gln Phe Gly Asn 130 135
140Asn Gly Glu Glu Ile Met Glu Asn Leu Ala Gln Gly Met Ile Leu
Leu145 150 155 160Lys Glu
Ala Tyr Val Asp Gln Pro Val Thr Leu Val Val Gly Ile Asp
165 170 175Ala Trp Lys Arg Ile Asn Arg
Glu Met Gln Gly His Pro Leu Ile Asn 180 185
190Arg Ile Gln Glu Leu Thr Gly Ser Lys Val Ile Tyr Ser Pro
Val Val 195 200 205Glu Gly Ala Leu
Leu Leu Pro Tyr Asp His Glu Asp Leu Glu Leu Thr 210
215 220Ile Gly Arg Asp Phe Ser Ile Gly Tyr Glu Tyr His
Asp Ala Lys Thr225 230 235
240Val Gln Leu Phe Ile Thr Glu Ser Leu Thr 245
250103251PRTDesulfovibrio 103Met Asp Ile Leu Lys Arg Asp Leu Ala Pro
Val Thr Ala Ala Ala Trp1 5 10
15Gln Ala Val Asp Ser Arg Ala Arg Gln Thr Leu Thr Thr Met Leu Ser
20 25 30Gly Arg Arg Val Val Asp
Val Ala Gly Pro Leu Gly Trp Glu Tyr Ala 35 40
45Ala Val Pro Leu Gly Arg Ile Glu Tyr Ala Lys Thr Gln Ser
Val Ser 50 55 60Gly Ile Thr Tyr Gly
Leu His Gln Val Lys Pro Leu Val Glu Val Lys65 70
75 80Val Pro Phe Thr Leu Asp Ile Ala Glu Ile
Asp Asn Ala Ala Arg Gly 85 90
95Gly Lys Asp Ile Asp Leu Ala Ala Leu Asp Glu Ala Ala Glu Lys Leu
100 105 110Ala Arg Phe Glu Glu
Glu Ala Leu Tyr His Gly Phe Ala Pro Ala Gly 115
120 125Ile Lys Gly Leu Ser Glu Val Ser Ser Gln Thr Arg
Leu Gln Val Ser 130 135 140Ser Asn Pro
Glu Asp Ile Ala Glu Lys Val Ser Lys Ala Leu Thr Ala145
150 155 160Leu Arg Lys Thr Ser Val Glu
Gly Pro Tyr Ala Leu Val Val Gly Pro 165
170 175Glu Leu Trp Val Ala Leu Ser Gly His Val Arg Gly
Tyr Pro Leu Ser 180 185 190Gln
Tyr Leu Glu Thr Met Leu Gly Gly Gln Val Ile Val Ser Pro Phe 195
200 205Ile Glu Glu Ala Tyr Leu Leu Ser Thr
Arg Gly Gly Asp Leu Glu Met 210 215
220Thr Leu Gly Gly Asp Ile Ala Ile Gly Tyr Ala Ser His Asp Thr Glu225
230 235 240Lys Val Ala Leu
Phe Phe Leu Glu Ser Phe Thr 245
250104251PRTHaliangium ochraceum 104Met Asp Leu Leu Lys Arg His Leu Ala
Pro Ile Val Pro Asp Ala Trp1 5 10
15Ser Ala Ile Asp Glu Glu Ala Lys Glu Ile Phe Gln Gly His Leu
Ala 20 25 30Gly Arg Lys Leu
Val Asp Phe Arg Gly Pro Phe Gly Trp Glu Tyr Ala 35
40 45Ala Val Asn Thr Gly Glu Leu Arg Pro Ile Asp Asp
Thr Pro Glu Asp 50 55 60Val Asp Met
Lys Leu Arg Gln Val Gln Pro Leu Ala Glu Val Arg Val65 70
75 80Pro Phe Thr Leu Asp Val Thr Glu
Leu Asp Ser Val Ala Arg Gly Ala 85 90
95Thr Asn Pro Asp Leu Asp Asp Val Ala Arg Ala Ala Glu Arg
Met Val 100 105 110Glu Ala Glu
Asp Ser Ala Ile Phe His Gly Trp Ala Gln Ala Gly Ile 115
120 125Lys Gly Ile Val Asp Ser Thr Pro His Glu Ala
Leu Ala Val Ala Ser 130 135 140Val Ser
Asp Phe Pro Arg Ala Val Leu Ser Ala Ala Asp Thr Leu Arg145
150 155 160Lys Ala Gly Val Thr Gly Pro
Tyr Ala Leu Val Leu Gly Pro Lys Ala 165
170 175Tyr Asp Asp Leu Phe Ala Ala Thr Gln Asp Gly Tyr
Pro Val Ala Lys 180 185 190Gln
Val Gln Arg Leu Val Val Asp Gly Pro Leu Val Arg Ala Asn Ala 195
200 205Leu Ala Gly Ala Leu Val Met Ser Met
Arg Gly Gly Asp Tyr Glu Leu 210 215
220Thr Val Gly Gln Asp Leu Ser Ile Gly Tyr Ala Phe His Asp Arg Ser225
230 235 240Lys Val Glu Leu
Phe Val Ala Glu Ser Phe Thr 245
250105252PRTSorangium cellulosum 105Met Asp Leu Leu Lys Arg Glu Leu Ala
Pro Ile Leu Pro Ala Ala Trp1 5 10
15Asp Leu Ile Asp His Glu Ala Thr Arg Val Leu Lys Leu His Leu
Ala 20 25 30Gly Arg Lys Val
Val Asp Phe Arg Gly Pro Phe Gly Trp Glu Val Ala 35
40 45Ala Val Asn Thr Gly Arg Leu Arg Ala Ile Glu Arg
Lys Glu Gly Pro 50 55 60Ala Val Ser
Ala Gly Val Arg Leu Val Arg Pro Leu Val Glu Phe Arg65 70
75 80Ala Pro Ile Arg Leu Glu Leu Ala
Glu Leu Asp Ala Val Gly Arg Gly 85 90
95Ala Gln Glu Pro Asn Ile Glu Asp Val Val Arg Ala Ala Glu
His Ala 100 105 110Ala Arg Phe
Glu Asp Gly Ala Ile Phe Asn Gly Leu Ala Ala Ala Gly 115
120 125Ile Glu Gly Ile Leu Glu Val Ala Pro His Lys
Pro Val Val Ile Pro 130 135 140Ala Pro
Glu Ala Trp Pro Arg Ala Val Ala Glu Ala Arg Glu Val Leu145
150 155 160Arg Ala Ala Gly Val Asp Gly
Pro Tyr Ala Leu Ala Leu Gly Pro Lys 165
170 175Ala Tyr Asp Glu Leu Ala Ala Ala Ala Glu Asp Gly
Tyr Pro Leu Arg 180 185 190Lys
His Ile Glu Gly Gln Leu Ile Asp Gly Pro Ile Val Trp Ala Pro 195
200 205Ala Leu Glu Gly Gly Val Leu Leu Ser
Thr Arg Gly Gly Asp Phe Glu 210 215
220Leu Thr Val Gly Glu Asp Leu Ser Ile Gly Tyr Asp Gly His Asp Arg225
230 235 240Gln Val Val Glu
Leu Phe Leu Thr Glu Ser Phe Thr 245
250106257PRTHungateiclostridium thermocellum 106Met Asp Phe Leu Ser Arg
Glu Gly Ser Pro Ile Ser Ala Glu Leu Trp1 5
10 15Glu Lys Ile Asp Glu Ala Val Val Ser Ala Ala Lys
Lys Ile Leu Thr 20 25 30Gly
Arg Arg Phe Ile Ser Ile Tyr Gly Pro Leu Gly Ala Gly Ile Gln 35
40 45Ala Ile Asn Val Asp Asn Ile Ser Glu
Leu Asp Glu Thr Glu Glu Asn 50 55
60Ile Ser Val Ile Arg Gly Arg Thr Tyr Arg His Ile Pro Leu Ile Asn65
70 75 80Glu Asp Phe Ser Leu
Leu Trp Arg Asp Leu Glu Phe Ser Glu Gln Met 85
90 95Gly Leu Pro Val Asp Leu Ser Ser Ala Ser Arg
Ala Ala Thr Gln Cys 100 105
110Ala Leu Arg Glu Asp Lys Leu Ile Phe Tyr Gly Asn Asp Glu Leu Gly
115 120 125Tyr Lys Gly Leu Leu Thr Glu
Asp Gly Ile Val Lys Phe Pro Ile Ser 130 135
140Asn Trp Ser Glu Gly Glu Asn Pro Phe Lys Asp Ile Ser Ile Gly
Leu145 150 155 160Ala Lys
Phe Ile Glu Asn Gly Ile Val Gly Arg Lys Ala Leu Val Val
165 170 175Ser Pro Asn Leu Phe Val Gln
Leu Gln Arg Ile Gln Pro Gly Thr Gly 180 185
190Thr Thr Glu Tyr Asp Arg Ile Asn Lys Leu Leu Asp Gly Asn
Ile Phe 195 200 205Ser Thr Pro Val
Leu Lys Asp Asp Lys Ala Val Leu Val Cys Ser Glu 210
215 220Pro Gln Asn Ile Asp Leu Val Ile Gly Gln Asp Met
Ile Thr Ser Tyr225 230 235
240Leu Glu Thr Lys Asn Leu Asn His Tyr Phe Arg Ile Met Glu Thr Ile
245 250
255Leu107251PRTNatranaerobius thermophilus 107Met Asn Leu Phe Lys Glu Gln
Leu Ala Pro Leu Thr Asn Ala Ala Trp1 5 10
15Asn Glu Ile Asn Asp Arg Ala Ala Gln Val Ile Lys Ser
Asn Leu Ser 20 25 30Thr Arg
Lys Val Phe Lys Ile Asn Gly Pro Lys Gly Leu Asp Tyr Pro 35
40 45Ala Val Ser Glu Gly Arg Leu Ser Glu Ile
Phe His Gly His Gln Gln 50 55 60Gly
Glu Val Lys Ala Gly Leu His Gln Val Lys Pro Leu Met Glu Thr65
70 75 80Arg Ile Thr Phe Lys Leu
Asp Arg Trp Glu Leu Asp Asn Ile Glu Arg 85
90 95Gly Ala Gln Asp Ile Asp Leu Glu Pro Leu Glu Asp
Ala Ala Arg Lys 100 105 110Ile
Ala Leu Phe Glu Glu Asn Ala Ile Tyr Asn Gly His Asn Asp Gly 115
120 125Gln Ile Pro Gly Leu Lys Thr Val Leu
Thr Gln Asp Leu Pro Leu Gly 130 135
140Asn Thr Gly Ser Glu Ile Met Glu Ser Ile Thr Arg Gly Ile Ile Thr145
150 155 160Leu Arg Lys Ala
Tyr Ile Ser Gln Asn Met Thr Leu Ile Val Gly Glu 165
170 175Glu Ala Trp Arg Lys Ile Asn Lys Glu Met
Ser Gly Glu Pro Leu Ile 180 185
190Glu Arg Ile His Glu Leu Thr Gly Ser Lys Val Val Ile Ser Pro Ile
195 200 205Val Asp Gly Ala Tyr Leu Val
Pro Tyr Asp His Asp Asp Leu Glu Leu 210 215
220Thr Ile Gly Leu Asp Phe Ser Ile Gly Tyr Glu His His Asp Glu
His225 230 235 240His Val
Gln Leu Phe Ile Thr Glu Ser Phe Thr 245
250108249PRTHalothermothrix orenii 108Met Val Asn Leu Lys Arg Ser Leu Ala
Pro Ile Thr Pro Asp Ala Trp1 5 10
15Glu Phe Ile Asp Lys Glu Ala Arg Arg Val Leu Lys Leu Lys Leu
Ser 20 25 30Ala Arg Lys Ala
Val Asp Phe Val Gly Pro Lys Gly Ile Lys Tyr Ala 35
40 45Ala Val Asn Thr Gly Arg Arg Thr Ala Leu Glu Asp
Lys Ala Glu Gly 50 55 60Ala Ser Ile
Phe Gln Arg Gln Val Leu Pro Leu Val Glu Val Glu Ile65 70
75 80Pro Phe Arg Leu His Leu Glu Glu
Leu Glu Ala Phe Val Arg Gly Ala 85 90
95Glu Asp Val Asn Ile Asp Asn Leu Leu Glu Ser Ala Asn Glu
Leu Ala 100 105 110Arg Ile Glu
Asn Lys Ala Ile Phe Phe Gly Met Asp Ser Ala Gly Ile 115
120 125Ser Gly Leu Val Asn Ser Ser Gly Gln Thr Leu
Asp Thr Pro Ala Thr 130 135 140Gly Leu
Ile Ser Ser Val Ala Glu Gly Ile Asn Asn Leu Val Lys Ala145
150 155 160Gly Val Asn Gly Pro Tyr Thr
Leu Leu Leu Gly Pro Glu Leu Tyr His 165
170 175Ser Leu Tyr Thr Arg Asn Asp Arg Gly Tyr Pro Leu
Glu Lys Arg Ile 180 185 190Ser
Asp Ile Ile Gly Gly Asp Ile Leu Phe Thr Pro Asp Leu Glu Gly 195
200 205Tyr Gly Leu Leu Leu Ser Lys Arg Gly
Gly Asp Phe Glu Leu Ile Val 210 215
220Gly Gln Asp Ile Ala Ile Gly Phe Ser Gly Gln Phe Gly Asp Glu Leu225
230 235 240Glu Phe Phe Leu
Leu Glu Ser Phe Thr 245109251PRTPetrotoga mobilis 109Met
Asp Phe Leu Lys Arg Glu Leu Ala Pro Ile Thr Glu Glu Ala Trp1
5 10 15Glu Glu Leu Asp Glu Arg Ala
Lys Glu Ile Phe Lys Asn Lys Leu Lys 20 25
30Ile Arg Pro Ile Ile Asp Val Glu Gly Pro Tyr Gly Trp Asp
Tyr Ser 35 40 45Ser Tyr Asn Leu
Gly Thr Asn Glu Leu Ile Glu Asn Pro Arg Asp Gly 50 55
60Leu Gly Trp Gly Ile Arg Gln Val Leu Pro Ile Val Glu
Ile Arg Asn65 70 75
80Pro Phe Val Leu Lys Gln Trp Glu Leu Asp Asn Ile Glu Arg Gly Leu
85 90 95Lys Thr Pro Asp Leu Glu
Gly Leu Glu Thr Ala Ala Lys Gln Leu Ala 100
105 110Ser Phe Glu Asn Lys Leu Ile Leu Lys Gly Ile Glu
Lys Ala Asn Ile 115 120 125Ile Gly
Leu Gln Thr Leu Ala Lys Gln Asn Ser Val Glu Ser Ser Lys 130
135 140Glu Ser Leu Lys Asp Phe Val Lys Ser Leu Phe
Glu Val Lys Lys Arg145 150 155
160Phe Met Glu Gln Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Lys
165 170 175Glu Leu Trp Gln
Asp Leu Phe Thr Met Asn Leu Ser Tyr Pro Leu Asp 180
185 190Leu Val Val Lys Glu Ile Ile Asp Ala Lys Val
Lys Pro Met His Glu 195 200 205Val
Asp Glu Ser Phe Val Val Ser Asn Arg Gly Gly Asp Phe Lys Leu 210
215 220Ile Leu Gly Gln Asp Ile Ser Leu Gly Tyr
Glu Ser Lys Phe Asp Glu225 230 235
240Gln Leu Lys Phe Phe Phe Thr Glu Ser Leu Thr
245 250110265PRTSulfurihydrogenibium sp. YO3AOP1 110Met
Glu Phe Leu Lys Arg Asn Glu Ala Pro Leu Ser Glu Ser Asp Trp1
5 10 15Glu Lys Ile Asp Lys Val Val
Val Glu Thr Ala Lys Arg Val Leu Val 20 25
30Gly Arg Arg Phe Ile Glu Ile Ser Gly Pro Tyr Asp Pro Ser
Val Gln 35 40 45Phe Val Pro Tyr
Asp Tyr Ile Glu Asp Gly Asn Ser Gly Ala Cys Gly 50 55
60Leu Phe Gly Glu Val Asp Cys Gly Val Val Lys Val Lys
Glu Arg Lys65 70 75
80Ile Leu Pro Leu Pro Ile Ile Tyr Lys Asp Phe Lys Ile His Trp Arg
85 90 95Asp Val Glu Ser Ser Lys
Lys Phe Asn Ile Pro Ile Asp Phe Ser Val 100
105 110Ala Ala Ala Ala Ala Ser Gln Val Ala Ile Ala Glu
Asp Arg Leu Ile 115 120 125Phe His
Gly Asp Ile Glu Thr Gly Phe Pro Gly Leu Leu Asn Val Glu 130
135 140Gly Lys Asn Ser Ile Ser Ile Ser Asp Trp Asn
Gln Thr Gly Glu Ala145 150 155
160Phe Lys Asp Ile Leu Asn Ala Ile Val Lys Leu Asn Glu Asn Gly Phe
165 170 175Tyr Asn Asn Phe
Ala Leu Val Leu Asn Pro Gln Asp Tyr Ala Met Leu 180
185 190Asn Arg Leu Tyr Gly Asn Ser Gly Ile Leu Glu
Ile Asp Gln Ile Lys 195 200 205Lys
Leu Phe Asp Val Gly Val Phe Thr Thr Pro Val Ile Pro Gln Phe 210
215 220Thr Ala Val Val Val Ser Thr Gly Ile Glu
Asn Leu Asp Leu Phe Ile225 230 235
240Ser Gln Asp Met Ile Thr Ser Tyr Leu Asn Tyr Asp Asn Met Asp
His 245 250 255Tyr Phe Arg
Val Phe Glu Ile Leu Ala 260
265111252PRTKocuria 111Met Asn Asn Leu His Arg Asp Leu Ala Pro Ile Ser
Ser Ala Ala Trp1 5 10
15Ala Asp Met Gly Asp Glu Ala Arg Arg Thr Phe Ala Ala Arg Ala Ala
20 25 30Ala Arg Arg Thr Val Asp Met
Pro Glu Pro Ala Gly Ala Glu Phe Ser 35 40
45Ala Leu Gly Thr Gly His Val Ser Arg Val Ala Ala Asp Thr Pro
Gly 50 55 60Val Glu Ala Leu Gln Arg
His Val Val Arg Val Val Glu Leu Arg Ala65 70
75 80Pro Phe Thr Leu Lys Arg Ser Asp Ile Asp Asp
Val Glu Arg Gly Ala 85 90
95Ala Asp Pro Asp Trp Gln Pro Val Lys Asp Ala Ala Val Ala Leu Ala
100 105 110Ser Ala Glu Asp Arg Thr
Val Phe Tyr Gly Ser Asp Ser Ala Gly Ile 115 120
125Gln Gly Ile Ala Pro Ala Ser Asp Asn Glu Arg Leu Ser Leu
Pro Gln 130 135 140Asp Val Arg Glu Phe
Pro Asn Ala Val Ala Lys Ala Lys Thr Glu Leu145 150
155 160Arg Leu Ala Gly Val Ala Gly Pro Tyr Asn
Leu Leu Leu Pro Ala Glu 165 170
175Leu Tyr Thr Glu Val Thr Glu Thr Thr Asp His Gly Tyr Pro Val His
180 185 190Glu His Val Ser Arg
Ile Leu Gly Glu Gly Ser Ile Ile Trp Ala Pro 195
200 205Ala Leu Asp Asp Ala Leu Leu Val Ser Ala Arg Gly
Gly Asp Tyr Glu 210 215 220Leu His Leu
Gly Gln Asp Ala Ala Ile Gly Tyr Thr Ser His Thr Ala225
230 235 240Glu Thr Val Glu Leu Tyr Leu
Arg Glu Thr Leu Thr 245
250112265PRTHydrogenobaculum 112Met Asn Phe Leu His Arg Glu Glu Ser Pro
Leu Thr Ala Gln Glu Trp1 5 10
15Gln Thr Ile Asp Asn Ile Val Val Asn Thr Ala Arg Asn His Leu Val
20 25 30Gly Arg Arg Phe Ile Glu
Leu Thr Gln Ala Leu Asp Pro Ala Ile Gln 35 40
45Ser Val Ala Tyr Asp Thr Ile Pro Ser Leu Asp Asn Gly Ala
Cys Gly 50 55 60Leu Phe Gly Glu Lys
Glu Cys Gly Ile Ala Lys Ile Lys Ser Arg Lys65 70
75 80Phe Leu Pro Ile Pro Gln Ile Tyr Lys Asp
Phe Lys Ile His Trp Arg 85 90
95Asp Ile Glu Thr Ser Arg Lys Leu Asn Ile Pro Leu Asp Val Ser Val
100 105 110Val Ala Leu Ala Thr
Arg Glu Val Ala Leu Ala Glu Asp Arg Phe Ile 115
120 125Phe His Gly Asp Ser Glu Ile Gly Tyr Pro Gly Leu
Leu Asn Val Glu 130 135 140Gly Arg Ser
Ile Ile Lys Asn Lys Asn Phe Asp Glu Glu Gly Gly Ile145
150 155 160Phe Lys Thr Ala Leu Ala Cys
Val Glu Thr Leu Val Glu Lys Gly Phe 165
170 175Ser Lys Asn Leu Ala Tyr Ile Leu Asn Pro Lys Asp
Tyr Thr Lys Ala 180 185 190Phe
Arg Ile Tyr Gly Asn Ser Gly Val Leu Glu Ile Thr His Ile Lys 195
200 205Glu Leu Phe Asp Val Gly Val Phe Thr
Ser His Ala Val Asp Glu Gly 210 215
220Lys Thr Ile Ala Val Ala Thr Gly Val Glu Asn Met Asp Ile Phe Leu225
230 235 240Val Gln Asp Met
Ile Ser Ala Phe Ile Asp Tyr Glu Asn Met Asp Tyr 245
250 255Tyr Phe Arg Val Phe Glu Ile Leu Ala
260 265113256PRTBrachyspira hyodysenteriae 113Met
Asp Tyr Leu Ala Arg Glu Ser Ser Pro Phe Glu Glu Ser Phe Trp1
5 10 15Gln Asn Ile Asp Lys Val Val
Val Glu Thr Ala Ser Arg Thr Leu Ile 20 25
30Gly Arg Arg Phe Leu Ser Ile Tyr Gly Pro Leu Gly Ala Gly
Ala Ile 35 40 45Ser Val Gln Tyr
Asp Lys Ser Asp Arg Glu Glu Val Phe Glu Asp Gly 50 55
60Phe Val Lys Thr Ser Gly Arg Lys Ser Val Glu Leu Pro
Gln Ile Tyr65 70 75
80Gln Asp Phe Thr Leu Leu Trp Arg Asp Leu Glu Asn Asn Ile Ser Asn
85 90 95Lys Leu Pro Leu Asp Leu
Ser Ile Val Ser Gln Ala Ala Gln Thr Leu 100
105 110Ala Asn Lys Glu Asp Asn Leu Ile Phe Asn Gly Asn
Asp Phe Leu Glu 115 120 125Leu Lys
Gly Ile Leu Asn Ala Glu Gly Ala Gln Lys Leu Gln Ile Ser 130
135 140Asp Trp Gly Gln Gly Glu Asn Pro Tyr Thr Asp
Ile Val Lys Ala Ile145 150 155
160Asn Met Ile Arg Glu Lys Gly Ile Val Gly Arg Phe Val Leu Cys Leu
165 170 175Ser Gln Ser Leu
Tyr Phe Asp Leu Gln Arg Ile Gln Gln Gly Thr Gly 180
185 190Met Thr Glu Ala Gln Arg Ile Ser Ser Met Ile
Gly Asn Leu Tyr Asn 195 200 205Val
Pro Val Ile Lys Gly Lys Lys Ala Ala Leu Ile Cys Ala Glu Pro 210
215 220Gln Tyr Met Asp Leu Ala Val Gly Ile Asp
Met Ser Thr Ala Tyr Leu225 230 235
240Glu Gln Lys Asp Leu Asn His Ser Phe Arg Ile Met Glu Thr Ile
Ile 245 250
255114251PRTDenitrovibrio acetiphilus 114Met Asn Leu Leu Arg Lys Asp Phe
Ala Pro Ile Gly Ser Ala Ala Trp1 5 10
15Asp Glu Ile Asn Thr Ile Ala Lys Glu Thr Leu Lys Ala Asn
Leu Ser 20 25 30Ala Arg Arg
Phe Ala Asp Val Glu Gly Pro Tyr Gly Ile Asn Phe Ala 35
40 45Ala Val Asn Leu Gly Arg Leu Lys Ile Ser Asp
Asn Lys Ser Pro Lys 50 55 60Asp Val
Val Tyr Gly Val Asn Thr Val Leu Pro Leu Val Glu Ala Arg65
70 75 80Ile Asn Phe Ser Leu Asp Ile
Trp Glu Leu Asp Asn Ile Asp Arg Gly 85 90
95Ala Lys Asp Ile Ala Leu Asp Asp Leu Ala Glu Ala Ala
Arg Lys Met 100 105 110Ala Asp
Phe Glu Glu Asn Ala Val Tyr Asn Gly Phe Lys Asp Ser Gly 115
120 125Ile Val Gly Leu Asn Gln Val Ala Ala Lys
Asn Arg Ile Asn Met Thr 130 135 140Leu
Asp Lys Asp Asn Leu Val Asp Ala Ile Ser Glu Ala Gln Gly Arg145
150 155 160Met Arg Lys Glu Gly Ile
Ala Ser Gly Ala Asn Leu Val Val Asn Pro 165
170 175Ala Leu Trp Gln Phe Leu Ala His Val Val Pro Gly
Gly Thr Leu Gly 180 185 190Asp
Thr Val Arg Arg Gln Ile Lys Gly Asp Ile Ile Tyr Ser Glu Thr 195
200 205Val Asp Gly Ala Leu Leu Val Ala Asp
Arg Glu Gly Asp Val Glu Leu 210 215
220Thr Thr Gly Gln Asp Phe Ala Ile Gly Tyr His Ser His Asp Ala Ser225
230 235 240Lys Val Asn Leu
Phe Leu Thr Glu Ser Phe Thr 245
250115249PRTThermanaerovibrio acidaminovorans 115Met Asp Val Leu Lys Arg
Gly Phe Ala Pro Ile Ser Gln Glu Ala Trp1 5
10 15Gly Val Leu Asp Gln Gln Ala Arg Val Ile Leu Arg
Glu Asn Leu Ser 20 25 30Ala
Arg Arg Phe Val Asp Val Glu Gly Pro Lys Gly Trp Asp Phe Pro 35
40 45Gly Phe Gly Thr Gly Arg Leu Val Leu
Pro Glu Gly Gln Gln Lys Gly 50 55
60Ala Val Arg Phe Gly Val Arg Gln Phe Gln Pro Met Ile Glu Thr Arg65
70 75 80Val Ser Phe Glu Ile
Ser Ile Trp Asp Leu Asp Asp Ile Ser Arg Gly 85
90 95Ala Val Asp Val Asp Leu Ser Ser Leu Glu Asp
Ala Ala Arg Lys Met 100 105
110Ala Glu Phe Glu Glu Arg Ala Ile Tyr His Gly Leu Asp Glu Gly Cys
115 120 125Ile Glu Gly Ile Val Lys Ser
Ala Gly Tyr Thr Ala Glu Leu Ser Val 130 135
140Ser Lys Ser Lys Asp Met Ile Met Gly Ile Ala Lys Gly Val Arg
Thr145 150 155 160Met Gly
Ala Ser Val Glu Gly Pro Phe Ala Leu Val Gly Gly Asp Lys
165 170 175Leu Phe Ala Ala Ile Asp Gly
Phe Ser Glu Pro Tyr Pro Met Arg Lys 180 185
190Asn Leu Ala Glu Leu Val Asp Lys Val Ile Tyr Ala Pro Ala
Leu Asp 195 200 205Gly Ala Leu Leu
Val Ser Leu Ala Gly Gly His Leu Gln Leu Thr Leu 210
215 220Gly Gln Asp Met Ser Leu Gly Tyr Glu Ala His Asp
Ser Thr Thr Val225 230 235
240Arg Leu Phe Phe Thr Glu Thr Phe Ala
245116257PRTEggerthella 116Met Asp Tyr Leu Ala Arg Glu Ser Ala Asp Leu
Ser Asp Gly Leu Trp1 5 10
15Asn Arg Ile Asp Glu Thr Val Ile Gly Thr Ala Arg Ala Gln Leu Thr
20 25 30Cys Arg Arg Phe Leu Lys Val
Phe Gly Pro Leu Gly Ala Gly Val Thr 35 40
45Thr Val Ala Val Asp Gly Val Asn Lys Glu Glu Val Leu Glu Asp
Gly 50 55 60Ile Gly Arg Ile Val Gly
Arg Thr Gln Leu Glu Leu Pro Leu Phe Tyr65 70
75 80Glu Asp Phe Thr Leu Leu Ser Arg Asp Met Glu
Tyr Ala Ala Gln Thr 85 90
95Gly Tyr Pro Leu Asp Leu Ser Val Ala Ile Ala Ala Ala Lys Lys Ala
100 105 110Ser Arg Arg Glu Asp Asp
Leu Ile Leu Asn Gly Ser Lys Ala Leu Gly 115 120
125Thr Asp Gly Leu Leu Thr Val Lys Gly Ser Ser Lys Ile Lys
Lys Ser 130 135 140Asp Trp Ser Gln Gly
Glu Asn Ser Phe Ala Asp Ile Thr Ala Gly Val145 150
155 160Ala Gln Leu Ala Lys Thr Gly Tyr Leu Gly
Arg Tyr Ala Leu Val Val 165 170
175Ser Pro Asp Leu Phe Leu Asp Leu Gln Arg Leu Gln Pro Asn Thr Gly
180 185 190Leu Leu Glu Ile Asp
Arg Ile Lys Lys Leu Ile Gly Asp Asn Val Tyr 195
200 205Met Thr Ser Val Met Gly Pro Gly Lys Ala Val Leu
Val Cys Ala Glu 210 215 220Pro Glu Tyr
Leu Asp Leu Ala Ile Gly Leu Asp Leu Ser Val Gly Tyr225
230 235 240Leu Glu Leu Ala Asp Phe Asn
His Thr Phe Arg Ile Met Glu Thr Ala 245
250 255Ala117246PRTPseudothermotoga 117Ala Asn Lys Tyr
Leu Met Gln Glu Asp Ala Pro Phe Asp Pro Lys Leu1 5
10 15Trp Gln Leu Phe Asn Glu Thr Met Thr Asp
Ile Ala Lys Ala Gln Leu 20 25
30Val Gly Arg Arg Ile Leu Ser Val Lys Gly Pro Phe Gly Leu Gly Leu
35 40 45Lys Gln Ile Ser Ile Thr Asp Val
Gln Ile Glu Pro Gly Val Phe Ser 50 55
60Asn Lys Thr Leu Pro Leu Phe Tyr Ile His Lys Thr Phe Asn Ile Ser65
70 75 80Lys Arg Asp Ile Ala
Ser Tyr Glu Arg Glu Gly Val Thr Leu Asp Leu 85
90 95Lys Asn Leu Ile Thr Ala Val Arg Glu Cys Ala
Thr Ile Glu Asp Arg 100 105
110Leu Ile Phe Glu Gly Ile Asn Ser His Gly Leu Val Ser Ala Pro Gly
115 120 125Thr Ile Ser Met Glu Leu Ser
Asp Trp Lys Asn Val Gly Gln Ala Ala 130 135
140Ser Asp Val Ile Glu Ala Val Thr Lys Leu Asp Glu Ala Gly Phe
His145 150 155 160Gly Pro
Tyr Leu Leu Ala Leu Ser Pro Asp Arg Tyr Asn Leu Leu Phe
165 170 175Arg Arg Tyr Glu Ser Gly Asn
Gln Thr Glu Tyr Glu His Leu Ser Met 180 185
190Ile Ile Lys Gly Ile Tyr Lys Ala Pro Val Leu Lys Asn Ser
Gly Val 195 200 205Leu Met Ser Asp
Ser Glu Ala Tyr Ala Ser Ile Ile Leu Gly Gln Asp 210
215 220Leu Ser Ile Gly Phe Ile Gly Pro Ala Glu Glu Arg
Phe Glu Phe Ser225 230 235
240Ile Ser Glu Ser Leu Ala 245118254PRTAnaeromyxobacter
sp. Fw109?5 118Thr Thr Trp Leu Asp Arg Glu Gly Ala Pro Phe Ala Gln Glu
Val Trp1 5 10 15Asp Arg
Ile Asp Ala Val Ala Arg Ser Ala Ala Asp Glu Val Arg Ala 20
25 30Gly Arg Arg Leu Leu Glu Val Val Gly
Pro Leu Gly Phe Gly Ala Arg 35 40
45Ala Gly Val Ala Glu Asp Leu Pro Leu Gly Glu Glu Pro Glu Gly Ala 50
55 60His Val His Val Pro Arg Val Arg Pro
Leu Pro Val Ile His Arg Thr65 70 75
80Phe Ala Leu Gly Ala Arg Ala Leu Glu Ala Asp Ala Ala Cys
Gly Glu 85 90 95Pro Leu
Val Leu Ser Glu Ala Ser Glu Ala Ala Arg Gln Ile Ala Arg 100
105 110Ala Glu Asp Arg Ile Val Phe Glu Gly
Leu Pro Arg Ala Gly Val Ser 115 120
125Gly Leu Leu Gly His Glu Gly Ala Val Glu Leu Pro Ala Gly Asp Trp
130 135 140Ser Asp Pro Ala Arg Val Ala
Asp Asp Leu Leu Gly Ala Leu Ala Lys145 150
155 160Leu Asp Glu Ala Gly Arg His Gly Pro Tyr Ala Leu
Ala Val Ser Pro 165 170
175Gly Arg Phe Tyr Gln Leu Leu Arg Pro Tyr Pro Gly Thr Ala Leu Thr
180 185 190Pro His Gln Gln Leu Gln
Pro Ala Phe Ala Gly Gly Ile Val Lys Ala 195 200
205Pro Ala Ile Gln Asp Gly Ala Val Ile Val Met Arg Thr Pro
Ser Gly 210 215 220Pro Arg Ile Leu Val
Gly Gln Glu Leu Ala Ala Ala Tyr Asp Gly Arg225 230
235 240Glu Gly Ile Phe His Gln Ile Ser Leu Val
Glu Ser Val Thr 245 250119265PRTMyxococcus
xanthus DK 1622 119Pro Asp Phe Leu Gly His Ala Glu Asn Pro Leu Arg Glu
Glu Glu Trp1 5 10 15Ala
Arg Leu Asn Glu Thr Val Ile Gln Val Ala Arg Arg Ser Leu Val 20
25 30Gly Arg Arg Ile Leu Asp Ile Tyr
Gly Pro Leu Gly Ala Gly Val Gln 35 40
45Thr Val Pro Tyr Asp Glu Phe Gln Gly Val Ser Pro Gly Ala Val Asp
50 55 60Ile Val Gly Glu Gln Glu Thr Ala
Met Val Phe Thr Asp Ala Arg Lys65 70 75
80Phe Lys Thr Ile Pro Ile Ile Tyr Lys Asp Phe Leu Leu
His Trp Arg 85 90 95Asp
Ile Glu Ala Ala Arg Thr His Asn Met Pro Leu Asp Val Ser Ala
100 105 110Ala Ala Gly Ala Ala Ala Leu
Cys Ala Gln Gln Glu Asp Glu Leu Ile 115 120
125Phe Tyr Gly Asp Ala Arg Leu Gly Tyr Glu Gly Leu Met Thr Ala
Asn 130 135 140Gly Arg Leu Thr Val Pro
Leu Gly Asp Trp Thr Ser Pro Gly Gly Gly145 150
155 160Phe Gln Ala Ile Val Glu Ala Thr Arg Lys Leu
Asn Glu Gln Gly His 165 170
175Phe Gly Pro Tyr Ala Val Val Leu Ser Pro Arg Leu Tyr Ser Gln Leu
180 185 190His Arg Ile Tyr Glu Lys
Thr Gly Val Leu Glu Ile Glu Thr Ile Arg 195 200
205Gln Leu Ala Ser Asp Gly Val Tyr Gln Ser Asn Arg Leu Arg
Gly Glu 210 215 220Ser Gly Val Val Val
Ser Thr Gly Arg Glu Asn Met Asp Leu Ala Val225 230
235 240Ser Met Asp Met Val Ala Ala Tyr Leu Gly
Ala Ser Arg Met Asn His 245 250
255Pro Phe Arg Val Leu Glu Ala Leu Leu 260
265120248PRTMethanoregula boonei 120Ala Asn Val Tyr Leu Gly Arg Asp Glu
Ala Pro Ile Gly Ala Glu Ser1 5 10
15Trp Lys Leu Ile Asp Asp Val Ala Val Gln Ala Ala Lys Gly Gln
Leu 20 25 30Ala Gly Arg Arg
Leu Leu Ala Ile Glu Gly Pro Tyr Gly Phe Gly Leu 35
40 45Lys Ala Ile Pro Leu Gly Asp Tyr Ala Leu Glu Asp
Gly Ile Ser Ala 50 55 60Ser Val Ser
Leu Pro Leu Ser Leu Ile Arg Thr Glu Phe Ser Leu Gly65 70
75 80Lys Arg Asp Leu Ala Ala Tyr Glu
Arg Asp His Leu Ile Leu Asp Thr 85 90
95Ala Pro Val Ala Cys Ala Ala Met Asp Ala Ala Ala Lys Glu
Asp Arg 100 105 110Ile Ile Phe
Asn Gly Leu Ala Gly Thr Pro Gly Leu Leu Asn Ala Glu 115
120 125Gly Ala Gly Ser Leu Thr Leu Ser Lys Trp Asp
Lys Val Gly Ala Ala 130 135 140Ala Asp
Gln Ile Ile Asp Ala Val Thr Lys Leu Asp Ala Ala Gly Phe145
150 155 160His Gly Pro Tyr Ser Leu Ala
Leu Ala Pro Ala Gln Tyr Asn Leu Leu 165
170 175Leu Arg Arg Tyr Pro Gln Gly Asp Gly Thr Glu Leu
Asp His Val Ser 180 185 190Ser
Ile Val Gly Asp Gly Val Ile Lys Ala Pro Val Leu Lys Lys Gly 195
200 205Gly Val Leu Val Ala Ser Gly Ser Gln
Tyr Ala Ser Val Ala Leu Gly 210 215
220Gln Asp Leu Ala Val Gly Tyr Asn Gly Pro Val Gly Asp Leu Leu Glu225
230 235 240Phe Gln Ile Tyr
Glu Ser Leu Ala 245121248PRTMethanosphaerula palustris
121Gly Glu Ser Tyr Leu Gly Arg Ser Asp Ala Pro Ile Thr Ala Glu Thr1
5 10 15Trp Thr Ile Ile Asp Thr
Thr Met Val Glu Ala Ala Lys Ser Met Leu 20 25
30Thr Gly Arg Arg Leu Leu His Leu Glu Gly Pro Tyr Gly
Leu Gly Leu 35 40 45Lys Ala Ile
Pro Leu Gln Asp Ser Val Ser Glu Gly Asn Leu Ile Arg 50
55 60Ser Gly Phe Ala Pro Val Asp Leu Ile Gln Thr Ser
Phe Ser Leu Ser65 70 75
80Lys Arg Asp Leu Ala Ala Tyr Glu Arg Asp Gly Met Leu Pro Asn Thr
85 90 95Ser Ala Val Ala Val Ala
Ala Ile Glu Ala Ala Arg Gln Glu Asp Ala 100
105 110Val Ile Phe Thr Gly Thr Asp Gln Val Lys Gly Leu
Met Asn Thr Gly 115 120 125Gly Ser
Gln Ser Val Lys Leu Ala Ser Trp Glu Lys Ile Gly Ala Ala 130
135 140Ala Asp Asp Leu Ile Lys Ala Val Thr Ala Leu
Asp Leu Ala Gly Phe145 150 155
160His Gly Pro Tyr Ala Leu Ala Leu Ser Pro Ala Arg Tyr Asn Leu Leu
165 170 175Phe Arg Arg Tyr
Pro Gln Gly Ser Thr Thr Glu Leu Glu His Leu Gln 180
185 190Gln Met Ile Thr Asp Gly Ile Phe Lys Ala Pro
Val Leu Lys Asp Gly 195 200 205Gly
Val Leu Ile Ala Thr Gly Gln Gln Tyr Ala Ala Ile Val Leu Gly 210
215 220Gln Asp Met Thr Ile Gly Phe Thr Gly Pro
Ser Lys Glu Ser Leu Asp225 230 235
240Phe Thr Ile Ser Glu Ser Leu Ala
245122265PRTBrevibacillus 122Met Asp Lys Leu Arg Lys Tyr Pro Asp Ser Pro
Leu Thr Thr Glu Glu1 5 10
15Trp Asn Gln Leu Asp Ala Thr Val Val Asp Met Ala Arg Arg Gln Leu
20 25 30Val Gly Arg Arg Phe Ile Asp
Ile Tyr Gly Pro Leu Gly Glu Gly Ile 35 40
45Gln Thr Ile Thr Asn Asp Val Tyr Glu Glu Ser Arg Phe Gly Gly
Leu 50 55 60Ser Leu Arg Gly Glu Ser
Leu Glu Met Thr Gln Pro Ser Arg Arg Val65 70
75 80Ser Met Thr Ile Pro Ile Leu Tyr Lys Asp Phe
Met Leu Tyr Trp Arg 85 90
95Asp Val Ala Gln Ala Arg Thr Leu Gly Met Pro Leu Asp Met Ser Ala
100 105 110Ala Ala Asn Ala Ala Ala
Gly Gly Ala Leu Met Glu Asp Asp Leu Ile 115 120
125Phe Asn Gly Ala Ala Glu Phe Asp Leu Pro Gly Leu Met Asn
Val Lys 130 135 140Gly Arg Leu Thr His
Leu Lys Ser Asp Trp Met Glu Ser Gly Asn Ala145 150
155 160Phe Ala Asp Ile Val Glu Ala Arg Asn Lys
Leu Leu Lys Met Gly His 165 170
175Ser Gly Pro Tyr Ala Leu Val Val Ser Pro Glu Leu Tyr Ser Leu Leu
180 185 190His Arg Val His Lys
Gly Thr Asn Val Leu Glu Ile Glu His Val Arg 195
200 205Asn Leu Val Thr Asp Gly Val Phe Gln Ser Pro Thr
Ile Lys Gly Arg 210 215 220Ser Gly Val
Leu Val Ala Thr Gly Arg His Asn Leu Asp Leu Ala Ile225
230 235 240Ala Glu Asp Phe Asp Ser Ala
Phe Leu Gly Asp Glu Gln Met Asn Ser 245
250 255Leu Phe Arg Val Tyr Glu Cys Val Val 260
265123251PRTMycobacterium 123Met Asn Asn Leu Tyr Arg Asp
Leu Ala Pro Val Thr Glu Ala Ala Trp1 5 10
15Ala Glu Ile Glu Leu Glu Ala Thr Arg Thr Phe Lys Arg
His Ile Ala 20 25 30Gly Arg
Arg Val Val Asp Val Ser Asp Pro Gly Gly Pro Val Thr Ala 35
40 45Ala Val Ser Thr Gly Arg Leu Ile Asp Val
Lys Ala Pro Thr Asp Gly 50 55 60Val
Ile Ala His Leu Arg Ala Ser Lys Pro Leu Val Arg Leu Arg Val65
70 75 80Pro Phe Thr Leu Ser Arg
Asn Glu Ile Asp Asp Val Glu Arg Gly Ser 85
90 95Gln Asp Ser Asp Trp Asp Pro Val Lys Ala Ala Ala
Lys Gln Leu Ala 100 105 110Phe
Val Glu Asp Arg Thr Ile Phe Glu Gly Tyr Gly Ala Ala Ser Ile 115
120 125Glu Gly Ile Arg Ser Ser Ser Ser Asn
Pro Pro Leu Thr Leu Pro Glu 130 135
140Asp Pro Arg Glu Ile Pro Asp Val Ile Thr Gln Ala Leu Ser Glu Leu145
150 155 160Arg Leu Ala Gly
Val Asp Gly Pro Tyr Ser Val Leu Leu Ala Ala Asp 165
170 175Val Tyr Thr Lys Val Ser Glu Thr Thr Glu
His Gly Tyr Pro Ile Arg 180 185
190Glu His Leu Asn Arg Leu Val Asp Gly Asp Ile Ile Trp Ala Pro Ala
195 200 205Ile Asp Gly Ala Phe Val Leu
Thr Thr Arg Gly Gly Asp Phe Asp Leu 210 215
220Gln Leu Gly Thr Asp Val Ala Ile Gly Tyr Thr Ser His Asp Ala
Asp225 230 235 240Thr Val
Gln Leu Tyr Leu Gln Glu Thr Leu Thr 245
250124251PRTTsukamurella paurometabola 124Met Asn Asn Leu Tyr Arg Asp Leu
Ala Pro Val Thr Ser Ala Ala Trp1 5 10
15Ser Glu Ile Glu Thr Glu Ala Thr Arg Thr Phe Lys Arg Asn
Ile Ala 20 25 30Gly Arg Arg
Val Val Asp Leu Gly Asp Pro Leu Gly Pro Thr Ala Ser 35
40 45Ala Val Gly Thr Gly His Leu Leu Glu Val Gly
Gly Pro Ala Glu Gly 50 55 60Val Gln
Ala His Leu Arg Asp Ser Arg Pro Leu Val Arg Leu Arg Val65
70 75 80Pro Phe Thr Leu Ser Arg Lys
Ala Ile Asp Ser Val Glu Arg Gly Ala 85 90
95Gln Asp Ala Asp Trp Asp Pro Val Lys Asp Ala Ala Arg
Ser Leu Ala 100 105 110Tyr Ala
Glu Asp Arg Ala Ile Phe Glu Gly Tyr Pro Asp Ala Ser Ile 115
120 125Pro Gly Ile Arg Thr Thr Ala Ala Gly Ser
Asp Leu Lys Leu Pro Asp 130 135 140Asp
Pro Arg Asp Ile Pro Asp Val Val Ser Gln Ala Leu Ser Asp Leu145
150 155 160Arg Leu Ala Gly Val Asp
Gly Pro Tyr Ser Val Leu Leu Ser Ala Asp 165
170 175Val Tyr Thr Arg Val Ser Glu Thr Ser Asp His Gly
Tyr Pro Val Arg 180 185 190Glu
His Leu Asn Arg Leu Val Asp Gly Asp Ile Ile Trp Ala Pro Ala 195
200 205Ile Asp Gly Ala Phe Val Leu Thr Thr
Arg Gly Gly Asp Phe Asp Leu 210 215
220Arg Leu Gly Thr Asp Val Glu Ile Gly Tyr Leu Ser His Thr Ala Asp225
230 235 240Thr Val Asp Leu
Tyr Leu Gln Glu Thr Phe Thr 245
250125251PRTRhodococcus 125Met Thr Asn Leu His Arg Asp Leu Ala Pro Ile
Ser Ala Ala Ala Trp1 5 10
15Ala Glu Ile Glu Glu Glu Ala Ser Arg Thr Phe Lys Arg His Val Ala
20 25 30Gly Arg Arg Val Val Asp Val
Glu Gly Pro Ser Gly Asp Asp Leu Ala 35 40
45Ala Ile Pro Leu Gly His Gln Val Pro Ile Asn Pro Leu Ala Asp
Gly 50 55 60Val Ile Ala His Ala Arg
Gln Ser Gln Pro Val Ile Glu Leu Arg Val65 70
75 80Pro Phe Thr Val Ser Arg Gln Ala Ile Asp Asp
Val Glu Arg Gly Ala 85 90
95Lys Asp Ser Asp Trp Gln Pro Val Lys Asp Ala Ala Lys Gln Ile Ala
100 105 110Phe Ala Glu Asp Arg Ala
Ile Phe Glu Gly Tyr Pro Ala Ala Ser Ile 115 120
125Thr Gly Val Arg Ala Ser Gly Ser Asn Pro Glu Leu Lys Leu
Pro Ile 130 135 140Asp Ala Lys Asp Tyr
Pro Glu Ala Ile Ser Gln Ala Ile Thr Ser Leu145 150
155 160Arg Leu Ala Gly Val Asn Gly Pro Tyr Ser
Leu Leu Leu Asn Ala Asp 165 170
175Ala Phe Thr Ala Ile Asn Glu Thr Ser Asp His Gly Tyr Pro Ile Arg
180 185 190Glu His Leu Arg Arg
Val Leu Asp Gly Glu Ile Ile Trp Ala Pro Ala 195
200 205Ile Asp Gly Ala Phe Leu Leu Ser Thr Arg Gly Gly
Asp Tyr Glu Leu 210 215 220His Leu Gly
Gln Asp Leu Ser Ile Gly Tyr Leu Ser His Asp Ala Asn225
230 235 240Ser Val Glu Leu Tyr Phe Gln
Glu Ser Met Thr 245
250126251PRTStreptomyces griseus 126Met Asn Asn Leu His Arg Glu Leu Ala
Pro Val Thr Pro Ser Ala Trp1 5 10
15Glu Glu Ile Glu Glu Glu Ala Arg Arg Thr Phe Arg Arg His Val
Ala 20 25 30Gly Arg Arg Val
Val Asp Val Ser Asp Pro Ala Gly Pro Thr Leu Ala 35
40 45Ala Val Gly Asp Gly His Leu Thr Asp Ile Asp Pro
Pro Thr Pro Asp 50 55 60Val Ala Ala
Arg Ala Arg Thr Ser Thr Pro Val Ile Glu Trp Arg Val65 70
75 80Pro Phe Thr Val Thr Arg Gln Ala
Val Asp Asp Val Glu Arg Gly Ser 85 90
95Ala Asp Ser Asp Trp Gln Pro Val Lys Asp Ala Ala Arg Thr
Cys Ala 100 105 110Phe Ala Glu
Asp Met Ala Ile Ile Asp Gly Tyr Gly Ala Ala Gly Ile 115
120 125Thr Gly Leu Arg Asp Gly Ser Ser His Asp Pro
Leu Pro Leu Pro Ala 130 135 140Asp Ala
Arg Asp Tyr Pro Val Ala Val Ser Gln Ala Val Thr Arg Leu145
150 155 160Arg Leu Ala Gly Val Asp Gly
Pro Tyr Arg Leu Leu Leu Gly Ala Asp 165
170 175Ala Phe Thr Glu Ala Ala Glu Thr Ser Asp His Gly
Tyr Pro Val Lys 180 185 190Thr
His Leu Ser Arg Leu Val Asp Asp Glu Ile Leu Trp Ala Pro Ala 195
200 205Val Lys Gly Gly Val Leu Leu Ser Thr
Arg Gly Gly Asp Phe Glu Leu 210 215
220Cys Leu Gly Gln Asp Leu Ser Ile Gly Tyr Ala Asp His Asp Ala Thr225
230 235 240Ser Val His Leu
Tyr Phe Gln Gln Ala Phe Thr 245
250127251PRTParaburkholderia phymatum 127Met Asn Asn Leu His Arg Glu Leu
Ala Pro Ile Ser Ser Glu Ala Trp1 5 10
15Ser Gln Ile Glu Glu Glu Val Ala Arg Thr Phe Lys Arg Ser
Val Ala 20 25 30Gly Arg Arg
Val Val Asp Val Lys Gly Pro Gly Gly Val Asp Leu Ser 35
40 45Gly Val Gly Thr Gly His Gln Ser Thr Ile Ala
Ala Pro His His Gly 50 55 60Val Ile
Ala Lys Leu Ser Glu Val Lys Ala Leu Val Gln Leu Thr Val65
70 75 80Pro Phe Glu Leu Ser Arg Asp
Ala Ile Asp Ala Val Glu Arg Gly Ala 85 90
95Asn Asp Ser Asp Trp Gln Ala Ala Lys Asp Ala Ala Lys
Glu Leu Ala 100 105 110Tyr Ala
Glu Asp Arg Ala Ile Phe Asp Gly Tyr Lys Ala Ala Gly Ile 115
120 125Val Gly Ile Arg Glu Gly Ser Ser Asn Thr
Ser Leu Ala Leu Pro Ala 130 135 140Asp
Val Ala Asp Tyr Pro Asn Ala Ile Gly Gly Ala Leu Gln Gln Leu145
150 155 160Arg Leu Ala Gly Val Asp
Gly Pro Tyr Ser Val Leu Leu Gly Ala Asp 165
170 175Ala Tyr Thr Ala Leu Gly Glu Ala Ser Asp Gln Gly
Tyr Pro Val Ile 180 185 190Glu
His Ile Lys Arg Ile Val Asn Gly Glu Ile Ile Trp Ala Pro Ala 195
200 205Leu Glu Gly Gly Ser Val Leu Ser Met
Arg Gly Gly Asp Tyr Glu Leu 210 215
220His Leu Gly Gln Asp Val Ser Ile Gly Tyr Gln Ser His Thr Asp Ser225
230 235 240Thr Val Arg Leu
Tyr Leu Arg Glu Thr Leu Thr 245
250128251PRTMethylocella silvestris 128Met Asn Asn Leu His Arg Glu Leu
Ala Pro Ile Ser Asp Ala Ala Trp1 5 10
15Ala Gln Ile Glu Glu Glu Thr Thr Arg Thr Leu Lys Arg Tyr
Leu Ala 20 25 30Gly Arg Arg
Val Val Asp Met Pro Gln Thr Gly Gly Val Ala Leu Ser 35
40 45Ala Val Gly Thr Gly His Leu Leu Ser Ile Ala
Ala Pro Ala Glu Gly 50 55 60Val Leu
Ala Arg Gln Arg Glu Val Lys Pro Leu Val Glu Leu Arg Val65
70 75 80Pro Phe Glu Leu Ser Arg Ala
Ala Ile Asp Asp Val Glu Arg Gly Ala 85 90
95Asp Asp Ser Asp Trp Gln Pro Ala Lys Asp Ala Ala Lys
Thr Ile Ala 100 105 110Phe Ala
Glu Asp Arg Ala Ile Phe Asp Gly Tyr Ala Asp Ala Ala Ile 115
120 125Thr Gly Val Arg Gln Gly Thr Ser Asn Pro
Ile Met Thr Leu Pro Ala 130 135 140Asp
Val Arg Asp Tyr Pro Asp Ala Ile Ala His Ala Leu Ser Gln Leu145
150 155 160Arg Leu Val Gly Val Asn
Gly Pro Tyr Ala Val Leu Phe Gly Ala Glu 165
170 175Ala Tyr Thr Ala Leu Ala Glu Thr Ser Asp His Gly
Phe Pro Val Leu 180 185 190Glu
His Val Lys Arg Leu Val Glu Asp Gln Ile Phe Trp Ala Pro Ala 195
200 205Ile Ala Gly Ala Phe Val Leu Thr Thr
Arg Gly Gly Asp Phe Glu Leu 210 215
220Thr Leu Gly Gln Asp Val Ser Ile Gly Tyr Leu Ser His Thr Ala Glu225
230 235 240Thr Val Gln Leu
Tyr Leu Gln Glu Ser Phe Thr 245
250129251PRTParacoccus denitrificans 129Met Asp Asn Leu His Arg Lys Leu
Ala Pro Ile Ser Asp Ala Ala Trp1 5 10
15Ala Gln Ile Glu Asp Glu Ala Ala Arg Thr Leu Lys Arg Tyr
Leu Gly 20 25 30Ala Arg Arg
Val Val Asp Val His Gly Pro Glu Gly Phe Gly Leu Ser 35
40 45Ala Val Gly Thr Gly His Leu Arg Pro Ala Thr
Ala Leu Ala Glu Gly 50 55 60Val Glu
Ser His Arg Arg Glu Val Asn Pro Leu Leu Glu Leu Arg Val65
70 75 80Pro Phe Thr Leu Thr Arg Ala
Ala Ile Asp Asp Val Ala Arg Gly Ser 85 90
95Asn Asp Ser Asp Trp Gln Pro Leu Lys Asp Ala Ala Arg
Lys Ile Ala 100 105 110Leu Ala
Glu Asp Arg Leu Val Phe Leu Gly His Gly Asp Ala Gly Ile 115
120 125Arg Gly Ile Leu Pro Glu Thr Ser Asn Pro
Ile Val Ala Leu Pro Ala 130 135 140Asn
Val Ala Asp Tyr Pro Glu Ala Val Ala Ser Ala Val Ser Glu Leu145
150 155 160Arg Leu Ala Gly Val Asn
Gly Pro Tyr Ala Leu Ile Leu Gly Thr Thr 165
170 175Ala Phe Thr Ala Ala Asn Gly Gly Ala Glu Asp Gly
Tyr Pro Val Leu 180 185 190Lys
His Leu Glu Arg Leu Val Asp Val Pro Val Val Trp Ser Gln Ala 195
200 205Leu Glu Gly Gly Ala Val Val Thr Thr
Arg Gly Gly Asp Phe Asp Leu 210 215
220Trp Leu Gly Gln Asp Ile Ser Ile Gly Tyr Leu Ser His Asp Ala Ala225
230 235 240Ser Val Thr Leu
Tyr Leu Gln Glu Ser Leu Thr 245
250130251PRTAgrobacterium 130Met Asn Asn Leu His Arg Gln Leu Ala Pro Ile
Ser Asp Ser Ala Trp1 5 10
15Ala Gln Ile Glu Glu Glu Ala Ser Arg Thr Leu Lys Arg His Leu Ala
20 25 30Ala Arg Arg Val Val Asp Val
Gln Asp Pro Gly Gly Val Glu Leu Ser 35 40
45Ala Val Gly Thr Gly His Leu Lys Pro Ile Pro Gly Pro Gly Asp
Gly 50 55 60Val Gln Thr Ala Leu Arg
Glu Val Lys Thr Leu Val Glu Leu Arg Val65 70
75 80Pro Phe Lys Leu Thr Arg Gln Ala Ile Asp Asp
Val Glu Arg Gly Ala 85 90
95Glu Asp Ser Asp Trp Ser Pro Val Lys Asp Ala Ala Arg Lys Ile Ala
100 105 110Phe Ala Glu Asp Arg Ser
Val Phe Asp Gly Tyr Ala Ala Ala Gly Ile 115 120
125Gln Gly Ile Arg Glu Gly Ser Ser Asn Pro Ile Leu Pro Leu
Pro Ser 130 135 140Asn Val Arg Gly Tyr
Pro Asp Ala Ile Ala Lys Ala Val Ser Gln Leu145 150
155 160Arg Leu Ala Gly Val Asn Gly Pro Tyr Ala
Leu Val Leu Gly Thr Glu 165 170
175Ala Tyr Thr Ala Ala Ser Gly Gly Ser Asp Asp Gly Tyr Pro Val Phe
180 185 190His His Ile Glu Arg
Val Val Asp Gly Gly Ile Ile Trp Ala Pro Ala 195
200 205Ile Glu Gly Gly Phe Val Leu Thr Thr Arg Gly Gly
Asp Phe Glu Leu 210 215 220Asp Ile Gly
Gln Asp Ile Ser Ile Gly Tyr Leu Ser His Ser Ser Thr225
230 235 240Val Val Glu Leu Tyr Leu Gln
Glu Thr Phe Thr 245 250131253PRTFrankia
131Met Asn His Leu Leu Arg Gly His Ala Pro Leu Ser Glu Glu Ala Trp1
5 10 15Lys Ala Val Asp Glu Glu
Ala Arg Ser Arg Leu Thr Thr Asn Leu Ala 20 25
30Ala Arg Lys Leu Ile Asp Phe Ala Gly Pro His Gly Trp
Ala Tyr Ser 35 40 45Ala Thr Pro
Ile Gly Arg Val Thr Ala Leu Gln Ala Pro Pro Gly Glu 50
55 60Gly Val Arg Ala Arg Leu Arg Arg Val Leu Pro Val
Met Glu Leu Arg65 70 75
80Ala Ala Phe Ser Ile Asp Arg Gly Glu Leu Asp Ala Ile Asp Arg Gly
85 90 95Ala Asp Asp Ile Asp Leu
Ser Ala Leu Glu Glu Ala Ala Arg Arg Val 100
105 110Ala Thr Thr Glu Asn Ser Val Val Phe His Gly Tyr
Ala Glu Ala Gly 115 120 125Ile Ile
Gly Ile Thr Glu Ala Ser Ser His Pro Val Leu Glu Leu Gly 130
135 140Ala Asp Thr Asp Ser Tyr Pro Arg Thr Val Ala
Lys Ala Val Ala Leu145 150 155
160Leu Arg Arg Ala Gly Ile Gly Gly Pro Tyr Gly Leu Ala Ile Asp Pro
165 170 175Asp Gly Tyr Thr
Ala Ile Leu Glu Ala Thr Glu His Gly Gly Tyr Leu 180
185 190Leu Leu Asn His Leu Lys Gln Ile Leu Asp Gly
Pro Val Val Arg Ala 195 200 205Pro
Gly Val Arg Gly Ala Val Val Leu Ser Gln Arg Gly Gly Asp Phe 210
215 220Ile Leu Glu Ser Gly Gln Asp Leu Ser Val
Gly Tyr Ser Ser His Thr225 230 235
240Ala Glu Glu Val Glu Leu Tyr Leu Glu Gln Ser Phe Ser
245 250132253PRTArtificial SequenceSynthetic
polypeptide 132Met Asp Asn Leu Lys Arg Glu Leu Ala Pro Leu Thr Glu Glu
Ala Trp1 5 10 15Ala Glu
Ile Asp Glu Glu Ala Arg Glu Thr Ala Lys Arg His Leu Ala 20
25 30Gly Arg Arg Val Val Asp Val Glu Gly
Pro Leu Gly Trp Gly Tyr Ser 35 40
45Ala Val Pro Leu Gly Arg Leu Glu Glu Ile Glu Gly Pro Ala Glu Gly 50
55 60Val Gln Ala Gly Val Arg Gln Val Leu
Pro Leu Pro Glu Leu Arg Val65 70 75
80Pro Phe Thr Leu Ser Arg Arg Asp Leu Asp Ala Val Glu Arg
Gly Ala 85 90 95Lys Asp
Leu Asp Leu Ser Pro Val Ala Glu Ala Ala Arg Lys Leu Ala 100
105 110Arg Ala Glu Asp Arg Leu Ile Phe Asn
Gly Tyr Ala Glu Ala Gly Ile 115 120
125Glu Gly Leu Leu Asn Ala Ser Gly Asn Leu Lys Leu Pro Leu Ser Ala
130 135 140Asp Pro Gly Asp Ile Pro Asp
Ala Ile Ala Glu Ala Leu Thr Lys Leu145 150
155 160Arg Glu Ala Gly Val Glu Gly Pro Tyr Ala Leu Val
Leu Ser Pro Asp 165 170
175Leu Tyr Thr Ala Leu Phe Arg Val Tyr Asp Gly Thr Gly Tyr Pro Glu
180 185 190Ile Glu His Ile Lys Glu
Leu Val Asp Gly Gly Val Ile Trp Ala Pro 195 200
205Ala Leu Asp Gly Gly Ala Val Leu Val Ser Thr Arg Gly Gly
Asp Phe 210 215 220Asp Leu Thr Leu Gly
Gln Asp Leu Ser Ile Gly Tyr Leu Ser His Asp225 230
235 240Ala Asp Asn Val Glu Leu Phe Leu Thr Glu
Ser Phe Thr 245 250133269PRTT. maritima
133Met Glu Phe Leu Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp1
5 10 15Gln Glu Ile Asp Asn Arg
Ala Arg Glu Ile Phe Lys Thr Gln Leu Tyr 20 25
30Gly Arg Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp
Glu Tyr Ala 35 40 45Ala His Pro
Leu Gly Glu Val Glu Val Leu Ser Asp Glu Asn Glu Val 50
55 60Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile
Glu Leu Arg Ala65 70 75
80Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys
85 90 95Pro Asn Val Asp Leu Ser
Ser Leu Glu Glu Thr Val Arg Lys Val Ala 100
105 110Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu
Lys Ser Gly Val 115 120 125Lys Gly
Leu Leu Ser Phe Glu Glu Arg Lys Ile Glu Cys Gly Ser Thr 130
135 140Pro Lys Asp Leu Leu Glu Ala Ile Val Arg Ala
Leu Ser Ile Phe Ser145 150 155
160Lys Asp Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr Asp Arg
165 170 175Trp Ile Asn Phe
Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys 180
185 190Arg Val Glu Glu Cys Leu Arg Gly Gly Lys Ile
Ile Thr Thr Pro Arg 195 200 205Ile
Glu Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe Lys Leu 210
215 220Ile Leu Gly Gln Asp Leu Ser Ile Gly Tyr
Glu Asp Arg Glu Lys Asp225 230 235
240Ala Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln Val Val
Asn 245 250 255Pro Glu Ala
Leu Ile Leu Leu Lys Phe Ser Gly Gly Ser 260
265134315PRTArtificial SequenceSynthetic polypeptide 134Met Gly Asn Asn
Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His1 5
10 15Pro Arg Ile Glu Asn Leu Tyr Phe Gln Gly
Gly Thr Ser Pro Asp Phe 20 25
30Leu Gly His Ala Glu Asn Pro Leu Arg Glu Glu Glu Trp Ala Arg Leu
35 40 45Asn Glu Thr Val Ile Gln Val Ala
Arg Arg Ser Leu Val Gly Arg Arg 50 55
60Ile Leu Asp Ile Tyr Gly Pro Leu Gly Ala Gly Val Gln Thr Val Pro65
70 75 80Tyr Asp Glu Phe Gln
Gly Val Ser Pro Gly Ala Val Asp Ile Val Gly 85
90 95Glu Gln Glu Thr Ala Met Val Phe Thr Asp Ala
Arg Lys Phe Lys Thr 100 105
110Ile Pro Ile Ile Tyr Lys Asp Phe Leu Leu His Trp Arg Asp Ile Glu
115 120 125Ala Ala Arg Thr His Asn Met
Pro Leu Asp Val Ser Ala Ala Ala Gly 130 135
140Ala Ala Ala Leu Cys Ala Gln Gln Glu Asp Glu Leu Ile Phe Tyr
Gly145 150 155 160Asp Ala
Arg Leu Gly Tyr Glu Gly Leu Met Thr Ala Asn Gly Arg Leu
165 170 175Thr Val Pro Leu Gly Asp Trp
Thr Ser Pro Gly Gly Gly Phe Gln Ala 180 185
190Ile Val Glu Ala Thr Arg Lys Leu Asn Glu Gln Gly His Phe
Gly Pro 195 200 205Tyr Ala Val Val
Leu Ser Pro Arg Leu Tyr Ser Gln Leu His Arg Ile 210
215 220Tyr Glu Lys Thr Gly Val Leu Glu Ile Glu Thr Ile
Arg Gln Leu Ala225 230 235
240Ser Asp Gly Val Tyr Gln Ser Asn Arg Leu Arg Gly Glu Ser Gly Val
245 250 255Val Val Ser Thr Gly
Arg Glu Asn Met Asp Leu Ala Val Ser Met Asp 260
265 270Met Val Ala Ala Tyr Leu Gly Ala Ser Arg Met Asn
His Pro Phe Arg 275 280 285Val Leu
Glu Ala Leu Leu Leu Arg Ile Lys His Pro Asp Ala Ile Cys 290
295 300Thr Leu Glu Gly Ala Gly Ala Thr Glu Arg
Arg305 310 315135320PRTArtificial
SequenceSynthetic polypeptide 135Met Gly Asn Asn Arg Pro Val Tyr Ile Pro
Gln Pro Arg Pro Pro His1 5 10
15Pro Arg Ile Glu Asn Leu Tyr Phe Gln Gly Gly Thr Ser Glu Phe Leu
20 25 30Lys Arg Ser Phe Ala Pro
Leu Thr Glu Lys Gln Trp Gln Glu Ile Asp 35 40
45Asn Arg Ala Arg Glu Ile Phe Lys Thr Gln Leu Tyr Gly Arg
Lys Phe 50 55 60Val Asp Val Glu Gly
Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro Leu65 70
75 80Gly Glu Val Glu Val Leu Ser Asp Glu Asn
Glu Val Val Lys Trp Gly 85 90
95Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu Arg Ala Thr Phe Thr Leu
100 105 110Asp Leu Trp Glu Leu
Asp Asn Leu Glu Arg Gly Lys Pro Asn Val Asp 115
120 125Leu Ser Ser Leu Glu Glu Thr Val Arg Lys Val Ala
Glu Phe Glu Asp 130 135 140Glu Val Ile
Phe Arg Gly Cys Glu Lys Ser Gly Val Lys Gly Leu Leu145
150 155 160Ser Phe Glu Glu Arg Lys Gly
Gly Gly Gly Gly Glu Asn Leu Tyr Phe 165
170 175Gln Gly His His His His His His Gly Gly Gly Gly
Gly Ile Glu Cys 180 185 190Gly
Ser Thr Pro Lys Asp Leu Leu Glu Ala Ile Val Arg Ala Leu Ser 195
200 205Ile Phe Ser Lys Asp Gly Ile Glu Gly
Pro Tyr Thr Leu Val Ile Asn 210 215
220Thr Asp Arg Trp Ile Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro225
230 235 240Leu Glu Lys Arg
Val Glu Glu Cys Leu Arg Gly Gly Lys Ile Ile Thr 245
250 255Thr Pro Arg Ile Glu Asp Ala Leu Val Val
Ser Glu Arg Gly Gly Asp 260 265
270Phe Lys Leu Ile Leu Gly Gln Asp Leu Ser Ile Gly Tyr Glu Asp Arg
275 280 285Glu Lys Asp Ala Val Arg Leu
Phe Ile Thr Glu Thr Phe Thr Phe Gln 290 295
300Val Val Asn Pro Glu Ala Leu Ile Leu Leu Lys Phe Ser Gly Gly
Ser305 310 315
320136331PRTArtificial SequenceSynthetic polypeptide 136Met Gly Asn Asn
Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His1 5
10 15Pro Arg Ile Glu Asn Leu Tyr Phe Gln Gly
Gly Thr Ser Glu Phe Leu 20 25
30Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp Gln Glu Ile Asp
35 40 45Asn Arg Ala Arg Glu Ile Phe Lys
Thr Gln Leu Tyr Gly Arg Lys Phe 50 55
60Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro Leu65
70 75 80Gly Glu Val Glu Val
Leu Ser Asp Glu Asn Glu Val Val Lys Trp Gly 85
90 95Leu Arg Lys Gly Gly Glu Asn Leu Tyr Phe Gln
Gly Gly Gly Ser Leu 100 105
110Pro Leu Ile Glu Leu Arg Ala Thr Phe Thr Leu Asp Leu Trp Glu Leu
115 120 125Asp Asn Leu Glu Arg Gly Lys
Pro Asn Val Asp Leu Ser Ser Leu Glu 130 135
140Glu Thr Val Arg Lys Val Ala Glu Phe Glu Asp Glu Val Ile Phe
Arg145 150 155 160Gly Cys
Glu Lys Ser Gly Val Lys Gly Leu Leu Ser Phe Glu Glu Arg
165 170 175Lys Gly Gly Gly Gly Gly Glu
Asn Leu Tyr Phe Gln Gly His His His 180 185
190His His His Gly Gly Gly Gly Gly Ile Glu Cys Gly Ser Thr
Pro Lys 195 200 205Asp Leu Leu Glu
Ala Ile Val Arg Ala Leu Ser Ile Phe Ser Lys Asp 210
215 220Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr
Asp Arg Trp Ile225 230 235
240Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys Arg Val
245 250 255Glu Glu Cys Leu Arg
Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu 260
265 270Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe
Lys Leu Ile Leu 275 280 285Gly Gln
Asp Leu Ser Ile Gly Tyr Glu Asp Arg Glu Lys Asp Ala Val 290
295 300Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln
Val Val Asn Pro Glu305 310 315
320Ala Leu Ile Leu Leu Lys Phe Ser Gly Gly Ser 325
330137996DNAArtificial SequenceSynthetic polynucleotide
137atggggaaca accgtcccgt ctacatccca cagccgcgcc ctccacatcc ccgtattgaa
60aacttgtatt ttcaaggtgg tacctccgag tttctgaaac gcagcttcgc cccgctgacc
120gagaagcagt ggcaggagat cgacaatcgc gcccgcgaga tcttcaagac acagctgtac
180ggtcgcaagt tcgtggacgt ggaaggcccg tacggctggg aatatgccgc acaccctctg
240ggtgaggtgg aggtgctgag cgacgagaac gaagtggtta agtggggtct gcgcaagggt
300ggtgaaaacc tgtatttcca aggtggtggt agcctgccgt taatcgaact gcgcgcaacc
360ttcaccctgg acctgtggga gctggacaac ctggagcgcg gcaagccgaa cgtggacctg
420agtagcctgg aggaaaccgt gcgtaaggtg gccgagtttg aggacgaagt gattttccgc
480ggctgcgaga agagcggcgt taagggtctg ctgagcttcg aagagcgcaa gggtggggga
540ggcggtgaaa acttgtattt tcaaggtcat catcaccacc atcatggtgg agggggcggc
600atcgagtgcg gcagcacccc gaaagatctg ctggaggcca tcgttcgcgc cctgagcatc
660ttcagtaagg acggcatcga gggcccgtac accctggtga ttaacaccga ccgttggatc
720aacttcctga aagaagaggc gggtcactac ccgctggaaa aacgcgtgga agagtgtctg
780cgcggcggca agatcatcac cacacctcgc atcgaagacg ccttagtggt tagcgagcgc
840ggcggcgact ttaagctgat cctgggccag gacctgagca tcggctatga ggaccgtgaa
900aaggacgccg tgcgtctgtt catcacagaa accttcacct tccaggtggt gaacccggaa
960gccctgatcc tgctgaagtt cagcggtgga tcctaa
996138331PRTArtificial SequenceSynthetic polypeptide 138Met Gly Asn Asn
Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His1 5
10 15Pro Arg Ile Glu Asn Leu Tyr Phe Gln Gly
Gly Thr Ser Glu Phe Leu 20 25
30Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp Gln Glu Ile Asp
35 40 45Asn Arg Ala Arg Glu Ile Phe Lys
Thr Gln Leu Tyr Gly Arg Lys Phe 50 55
60Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala Ala His Pro Leu65
70 75 80Gly Glu Val Glu Val
Leu Ser Asp Gly Gly Glu Asn Leu Tyr Phe Gln 85
90 95Gly Gly Gly Glu Asn Glu Val Val Lys Trp Gly
Leu Arg Lys Ser Leu 100 105
110Pro Leu Ile Glu Leu Arg Ala Thr Phe Thr Leu Asp Leu Trp Glu Leu
115 120 125Asp Asn Leu Glu Arg Gly Lys
Pro Asn Val Asp Leu Ser Ser Leu Glu 130 135
140Glu Thr Val Arg Lys Val Ala Glu Phe Glu Asp Glu Val Ile Phe
Arg145 150 155 160Gly Cys
Glu Lys Ser Gly Val Lys Gly Leu Leu Ser Phe Glu Glu Arg
165 170 175Lys Gly Gly Gly Gly Gly Glu
Asn Leu Tyr Phe Gln Gly His His His 180 185
190His His His Gly Gly Gly Gly Gly Ile Glu Cys Gly Ser Thr
Pro Lys 195 200 205Asp Leu Leu Glu
Ala Ile Val Arg Ala Leu Ser Ile Phe Ser Lys Asp 210
215 220Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr
Asp Arg Trp Ile225 230 235
240Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys Arg Val
245 250 255Glu Glu Cys Leu Arg
Gly Gly Lys Ile Ile Thr Thr Pro Arg Ile Glu 260
265 270Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe
Lys Leu Ile Leu 275 280 285Gly Gln
Asp Leu Ser Ile Gly Tyr Glu Asp Arg Glu Lys Asp Ala Val 290
295 300Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln
Val Val Asn Pro Glu305 310 315
320Ala Leu Ile Leu Leu Lys Phe Ser Gly Gly Ser 325
3301399PRTArtificial SequenceSynthetic polypeptide 139Glu
Ile Glu Gly Pro Ala Glu Gly Val1 514011PRTArtificial
SequenceSynthetic polypeptide 140Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly
Gly1 5 1014113PRTArtificial
SequenceSynthetic polypeptide 141Leu Asn Ala Ser Gly Asn Leu Lys Leu Pro
Leu Ser Ala1 5 101429PRTArtificial
SequenceSynthetic polypeptide 142Gln Ala Gly Val Arg Gln Val Leu Pro1
51435PRTArtificial SequenceSynthetic polypeptide 143Gly Gly Gly
Gly Gly1 5
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